Lipid comprising long chain polyunsaturated fatty acids

ABSTRACT

The present invention relates to extracted plant lipid or microbial lipid comprising docosahexaenoic acid, and/or docosapentaenoic acid, and processes for producing the extracted lipid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/406,099, filed Jan. 13, 2017, now allowed, which is a divisional ofU.S. application Ser. No. 14/575,756, filed Dec. 18, 2014, now U.S. Pat.No. 9,725,399, issued Aug. 8, 2017, claiming priority of AustralianPatent Applications Nos. 2014902471, filed Jun. 27, 2014 and 2013905033,filed Dec. 18, 2013, the entire contents of each of which are herebyincorporated by reference into the subject application.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“181214_87180-AA_Substitute_Sequence_Listing_CAS.txt”, which is 173kilobytes in size, and which was created Dec. 14, 2018 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Dec. 14, 2018 aspart of this application.

FIELD OF THE INVENTION

The present invention relates to lipid comprising docosahexaenoic acidand/or docosapentaenoic acid, obtained from plant cells or microbialcells, and processes for producing and using the lipid.

BACKGROUND OF THE INVENTION

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) are now widelyrecognized as important compounds for human and animal health. Thesefatty acids may be obtained from dietary sources or by conversion oflinoleic (LA, 18:2ω6) or α-linolenic (ALA, 18:3ω3) fatty acids, both ofwhich are regarded as essential fatty acids in the human diet. Whilehumans and many other vertebrate animals are able to convert LA or ALA,obtained from plant sources to C22 they carry out this conversion at avery low rate. Moreover, most modern societies have imbalanced diets inwhich at least 90% of polyunsaturated fatty acids (PUFA) are of the ω6fatty acids, instead of the 4:1 ratio or less for ω6:ω3 fatty acids thatis regarded as ideal (Trautwein, 2001). The immediate dietary source ofLC-PUFAs such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoicacid (DHA, 22:6ω3) for humans is mostly from fish or fish oil. Healthprofessionals have therefore recommended the regular inclusion of fishcontaining significant levels of LC-PUFA into the human diet.Increasingly, fish-derived LC-PUFA oils are being incorporated into foodproducts and in infant formula, for example, However, due to a declinein global and national fisheries, alternative sources of thesebeneficial health-enhancing oils are needed.

Flowering plants, in contrast to animals, lack the capacity tosynthesise polyunsaturated fatty acids with chain lengths longer than 18carbons. In particular, crop and horticultural plants along with otherangiosperms do not have the enzymes needed to synthesize the longerchain ω3 fatty acids such as EPA, docosapentaenoic acid (DPA, 22:50) andDHA that are derived from ALA. An important goal in plant biotechnologyis therefore the engineering of crop plants which produce substantialquantities of LC-PUFA, thus providing an alternative source of thesecompounds.

LC-PUFA Biosynthesis Pathways

Biosynthesis of LC-PUFAs in organisms such as microalgae, mosses andfungi usually occurs as a series of oxygen-dependent desaturation andelongation reactions (FIG. 1). The most common pathway that produces EPAin these organisms includes a Δ6-desaturation, Δ6-elongation andΔ5-desaturation (termed the Δ6-desaturation pathway) whilst a lesscommon pathway uses a Δ9-elongation, Δ8-desaturation and Δ5-desaturation(termed the Δ9-desaturation pathway). These consecutive desaturation andelongation reactions can begin with either the ω6 fatty acid substrateLA, shown schematically as the upper left part of FIG. 1 (ω6) or the ω3substrate ALA through to EPA, shown as the lower right part of FIG. 1(ω3). If the initial Δ6-desaturation is performed on the ω6 substrateLA, the LC-PUFA product of the series of three enzymes will be the ω6fatty acid ARA. LC-PUFA synthesising organisms may convert ω6 fattyacids to ω3 fatty acids using an ω3-desaturase, shown as theΔ17-desaturase step in FIG. 1 for conversion of arachidonic acid (ARA,20:4ω6) to EPA. Some members of the ω3-desaturase family can act on avariety of substrates ranging from LA to ARA. Plant ω3-desaturases oftenspecifically catalyse the Δ15-desaturation of LA to ALA, while fungaland yeast ω3-desaturases may be specific for the Δ17-desaturation of ARAto EPA (Pereira et al., 2004a; Zank et al., 2005). Some reports suggestthat non-specific ω3-desaturases may exist which can convert a widevariety of ω6 substrates to their corresponding ω3 products (Zhang etal., 2008).

The conversion of EPA to DHA in these organisms occurs by aΔ5-elongation of EPA to produce DPA, followed by a Δ4-desaturation toproduce DHA (FIG. 1). In contrast, mammals use the so-called “Sprecher”pathway which converts DPA to DHA by three separate reactions that areindependent of a Δ4-desaturase (Sprecher et al., 1995).

The front-end desaturases generally found in plants, mosses, microalgae,and lower animals such as Caenorhabditis elegans predominantly acceptfatty acid substrates esterified to the sn-2 position of aphosphatidylcholine (PC) substrate. These desaturases are thereforeknown as acyl-PC, lipid-linked, front-end desaturases (Domergue et al.,2003). In contrast, higher animal front-end desaturases generally acceptacyl-CoA substrates where the fatty acid substrate is linked to CoArather than PC (Domergue et al., 2005). Some microalgal desaturases andone plant desaturase are known to use fatty acid substrates esterifiedto CoA (Table 2).

Each PUFA elongation reaction consists of four steps catalysed by amulti-component protein complex: first, a condensation reaction resultsin the addition of a 2C unit from malonyl-CoA to the fatty acid,resulting in the formation of a β-ketoacyl intermediate. This is thenreduced by NADPH, followed by a dehydration to yield an enoylintermediate. This intermediate is finally reduced a second time toproduce the elongated fatty acid. It is generally thought that thecondensation step of these four reactions is substrate specific whilstthe other steps are not. In practice, this means that native plantelongation machinery is capable of elongating PUFA providing that thecondensation enzyme (typically called an ‘elongase’) specific to thePUFA is introduced, although the efficiency of the native plantelongation machinery in elongating the non-native PUFA substrates may below. In 2007 the identification and characterisation of the yeastelongation cycle dehydratase was published (Denic and Weissman, 2007).

PUFA desaturation in plants, mosses and microalgae naturally occurs tofatty acid substrates predominantly in the acyl-PC pool whilstelongation occurs to substrates in the acyl-CoA pool. Transfer of fattyacids from acyl-PC molecules to a CoA carrier is performed byphospholipases (PLAs) whilst the transfer of acyl-CoA fatty acids to aPC carrier is performed by lysophosphatidyl-choline acyltransferases(LPCATs) (FIG. 9) (Singh et al., 2005).

Engineered Production of LC-PUFA

Most LC-PUFA metabolic engineering has been performed using the aerobicΔ6-desaturation/elongation pathway. The biosynthesis of γ-linolenic acid(GLA, 18:3ω6) in tobacco was first reported in 1996 using aΔ6-desaturase from the cyanobacterium Synechocystis (Reddy and Thomas,1996). More recently, GLA has been produced in crop plants such assafflower (73% GLA in seedoil, WO 2006/127789) and soybean (28% GLA;Sato et al., 2004). The production of LC-PUFA such as EPA and DHAinvolves more complicated engineering due to the increased number ofdesaturation and elongation steps involved. EPA production in a landplant was first reported by Qi et al. (2004) who introduced genesencoding a Δ9-elongase from Isochrysis galbana, a Δ8-desaturase fromEuglena gracilis and a Δ5-desaturase from Mortierella alpina intoArabidopsis yielding up to 3% EPA. This work was followed by Abbadi etal. (2004) who reported the production of up to 0.8% EPA in flax seedusing genes encoding a Δ6-desaturase and Δ6-elongase from Physcomitrellapatens and a Δ5-desaturase from Phaeodactylum tricornutum.

The first report of DHA production was in WO 04/017467 where theproduction of 3% DHA in soybean embryos is described, but not seed, byintroducing genes encoding the Saprolegnia diclina Δ6-desaturase,Mortierella alpina Δ6-desaturase, Mortierella alpina Δ5-desaturase,Saprolegnia diclina Δ4-desaturase, Saprolegnia diclina Δ17-desaturase,Mortierella alpina Δ6-elongase and Pavlova lutheri Δ5-elongase. Themaximal EPA level in embryos also producing DHA was 19.6%, indicatingthat the efficiency of conversion of EPA to DHA was poor (WO2004/071467). This finding was similar to that published by Robert etal. (2005), where the flux from EPA to DHA was low, with the productionof 3% EPA and 0.5% DHA in Arabidopsis using the Danio rerioΔ5/6-desaturase, the Caenorhabditis elegans Δ6-elongase, and the Pavlovasalina Δ5-elongase and Δ4-desaturase. Also in 2005, Wu et al. publishedthe production of 25% ARA, 15% EPA, and 1.5% DHA in Brassica junceausing the Pythium irregulare Δ6-desaturase, a ThraustochytridΔ5-desaturase, the Physcomitrella patens Δ6-elongase, the Calendulaofficianalis Δ12-desaturase, a Thraustochytrid Δ5-elongase, thePhytophthora infestans Δ17-desaturase, the Oncorhyncus mykiss LC-PUFAelongase, a Thraustochytrid Δ4-desaturase and a Thraustochytrid LPCAT(Wu et al., 2005). Summaries of efforts to produce oil-seed crops whichsynthesize ω3 LC-PUFAs is provided in Venegas-Caleron et al. (2010) andRuiz-Lopez et al. (2012). As indicated by Ruiz-Lopez et al. (2012),results obtained to date for the production of DHA in transgenic plantshas been no where near the levels seen in fish oils. More recently,Petrie et al (2012) reported the production of about 15% DHA inArabidopsis thaliana seeds, and WO2013/185184 reported the production ofcertain scedoils having between 7% and 20% DHA. However, there are noreports of production of plant oils having more than 20% DHA.

There are no reports of the production of DPA in recombinant cells tosignificant levels without concomitant production of DHA. Indeed, thepresent inventors are unaware of any published suggestion or motivationto produce DPA in recombinant cells without production of DHA.

There therefore remains a need for more efficient production of LC PUFAin recombinant cells, in particular of DHA or DPA in seeds of oilseedplants.

SUMMARY OF THE INVENTION

The present inventors have identified methods and plants for producinglipid with high levels of DHA and/or DPA. As described in WO2013/185184,the present inventors have previously disclosed extracted plant lipid,and plants and plant parts for producing such lipid, comprising DHA inthe total fatty acid content of the extracted lipid of between 7% and20%. An upper limited of 20% was defined because at the time it wasconsidered a maximal amount of DHA which could be produced in plants.However, as described herein, the inventors were surprised to find thatlevels of DHA in the total fatty acid content greater than 20% can beobtained. The inventors also found plant lipid, and plant parts andplants for producing lipid comprising DPA in the total fatty acidcontent of the extracted lipid of between 7% and 35%, particularly inthe absence of DHA.

Accordingly, in a first aspect the present invention provides extractedplant lipid, comprising fatty acids in an esterified form, the fattyacids comprising oleic acid, palmitic acid, ω6 fatty acids whichcomprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenicacid (ALA) and docosahexaenoic acid (DHA), and optionally one or more ofstearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoicacid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA inthe total fatty acid content of the extracted lipid is between 20.1% and30% or between 20.1% and 35%, preferably between 30% and 35%.

In another aspect, the present invention provides extracted plant lipid,comprising fatty acids in an esterified form, the fatty acids comprisingoleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid(LA), ω3 fatty acids which comprise α-linolenic acid (ALA) anddocosahexaenoic acid (DHA), and optionally one or more of stearidonicacid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) andeicosatetraenoic acid (ETA), wherein the level of palmitic acid in thetotal fatty acid content of the extracted lipid is between about 2% and16%, and wherein the level of myristic acid (C14:0) in the total fattyacid content of the extracted lipid, if present, is less than 1%, andwherein the level of DHA in the total fatty acid content of theextracted lipid is between 20.1% and 30% or between 20.1% and 35%,preferably between 30% and 35%.

In another aspect, the invention provides extracted lipid, preferablyextracted plant lipid or extracted microbial lipid, comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA) and docosapentaenoicacid (DPA), and optionally one or more of stearidonic acid (SDA),eicosapentaenoic acid (EPA), and eicosatetraenoic acid (ETA), whereinthe level of DPA in the total fatty acid content of the extracted lipidis between about 7% and 35%. In embodiments of this aspect, the level ofDPA in the total fatty acid content of the extracted lipid is about 7%,about 8%, about 9%, about 10%, about 12%, about 15%, about 18%, about20%, about 22%, about 24%, about 26%, about 28%, about 30%, betweenabout 7% and about 28%, between about 7% and about 25%, between about10% and 35%, between about 10% and about 30%, between about 10% andabout 25%, between about 10% and about 22%, between about 14% and 35%,between about 16% and 35%, between about 16% and about 30%, betweenabout 16% and about 25%, or between about 16% and about 22%.

In an embodiment of the above aspect, DHA is present at a level of lessthan 0.5% of the total fatty acid content of the extracted lipid andmore preferably is absent from the total fatty acid content of thelipid.

In another aspect, the invention provides extracted lipid, preferablyextracted plant lipid or extracted microbial lipid, comprising fattyacids in an esterified form, the fatty acids comprising docosapentaenoicacid (DPA) and/or docosahexaenoic acid (DHA), wherein at least 35% ofthe DPA and/or DHA esterified in the form of triacylglycerol (TAG) isesterified at the sn-2 position of the TAG. In an embodiment, theextracted lipid is further characterised by one or more or all of (i) itcomprises fatty acids comprising oleic acid, palmitic acid, ω6 fattyacids which comprise linoleic acid (LA), ω3 fatty acids which compriseα-linolenic acid (ALA) and optionally one or more of stearidonic acid(SDA), eicosapentaenoic acid (EPA), and eicosatetraenoic acid (ETA),(ii) at least about 40%, at least about 45%, at least about 48%, between35% and about 60%, or between 35% and about 50%, of the DPA and/or DHAesterified in the form of triacylglycerol (TAG) is esterified at thesn-2 position of the TAG, and (iii) the level of DPA and/DHA in thetotal fatty acid content of the extracted lipid is between about 1% and35%, or between about 7% and 35% or between about 20.1% and 35%. Inembodiments of this aspect, the level of DPA and/or DHA in the totalfatty acid content of the extracted lipid is about 7%, about 8%, about9%, about 10%, about 12%, about 15%, about 18%, about 20%, about 22%,about 24%, about 26%, about 28%, about 30%, between about 7% and about28%, between about 7% and about 25%, between about 10% and 35%, betweenabout 10% and about 30%, between about 10% and about 25%, between about10% and about 22%, between about 14% and 35%, between about 16% and 35%,between about 16% and about 30%, between about 16% and about 25%, orbetween about 16% and about 22%. In preferred embodiments, the extractedlipid is characterised by (i) and (ii), (i) and (iii) or (ii) and (iii),more preferably all of (i), (ii) and (iii). Preferably, the extractedlipid is further characterised by a level of palmitic acid in the totalfatty acid content of the extracted lipid which is between about 2% and16%, and a level of myristic acid (C14:0) in the total fatty acidcontent of the extracted lipid, if present, is less than 1%.

Embodiments of each of the four above aspects are described in furtherdetail below. As the skilled person would understand, any embodimentsdescribed which are broader than the corresponding feature in an aboveaspect do not apply to that aspect.

In an embodiment, the extracted lipid has one or more of the followingfeatures

-   -   i) the level of palmitic acid in the total fatty acid content of        the extracted lipid is between about 2% and 18%, between about        2% and 16%, between about 2% and 15%, or between about 3% and        about 10%,    -   ii) the level of myristic acid (C14:0) in the total fatty acid        content of the extracted lipid is less than 6%, less than 3%,        less than 2%, less than 1%, or about 0.1%,    -   iii) the level of oleic acid in the total fatty acid content of        the extracted lipid is between about 1% and about 30%, between        about 3% and about 30%, between about 6% and about 30%, between        1% and about 20%, between about 30% and about 60%, about 45% to        about 60%, about 30%, or between about 15% and about 30%,    -   iv) the level of linoleic acid (LA) in the total fatty acid        content of the extracted lipid is between about 4% and about        35%, between about 4% and about 20%, between about 4% and about        17%, or between about 5% and about 10%,    -   v) the level of α-linolenic acid (ALA) in the total fatty acid        content of the extracted lipid is between about 4% and about        40%, between about 7% and about 40%, between about 10% and about        35%, between about 20% and about 35%, between about 4% and 16%,        or between about 2% and 16%,    -   vi) the level of γ-linolenic acid (GLA) in the total fatty acid        content of the extracted lipid is less than 4%, less than about        3%, less than about 2%, less than about 1%, less than about        0.5%, between 0.05% and about 7%, between 0.05% and about 4%,        between 0.05% and about 3%, or between 0.05% and about 2%,    -   vii) the level of stearidonic acid (SDA) in the total fatty acid        content of the extracted lipid is less than about 10%, less than        about 8%, less than about 7%, less than about 6%, less than        about 4%, less than about 3%, between about 0.05% and about 7%,        between about 0.05% and about 6%, between about 0.05% and about        4%, between about 0.05% and about 3%, between about 0.05% and        about 10%, or between 0.05% and about 2%,    -   viii) the level of eicosatetraenoic acid (ETA) in the total        fatty acid content of the extracted lipid is less than about 6%,        less than about 5%, less than about 4%, less than about 1%, less        than about 0.5%, between 0.05% and about 6%, between 0.05% and        about 5%, between 0.05% and about 4%, between 0.05% and about        3%, or between 0.05% and about 2%,    -   ix) the level of eicosatrienoic acid (ETrA) in the total fatty        acid content of the extracted lipid is less than 4%, less than        about 2%, less than about 1%, between 0.05% and 4%, between        0.05% and 3%, or between 0.05% and about 2%, or between 0.05%        and about 1%,    -   x) the level of eicosapentaenoic acid (EPA) in the total fatty        acid content of the extracted lipid is between 4% and 15%, less        than 4%, less than about 3%, less than about 2%, between 0.05%        and 10%, between 0.05% and 5%, between 0.05% and about 3%, or        between 0.05% and about 2%,    -   xi) if the level of DHA in the total fatty acid content of the        extracted lipid is between 20.1% and 35%, the level of        docosapentaenoic acid (DPA) in the total fatty acid content of        the extracted lipid is less than 4%, less than about 3%, less        than about 2%, between 0.05% and 8%, between 0.05% and 5%,        between 0.05% and about 3%, between 5% and 15%, between 5% and        10%, or between 0.05% and about 2%,    -   xii) the level of DHA in the total fatty acid content of the        extracted lipid is about 22%, about 24%, about 26%, about 28%,        about 31%, between 20.1% and 29%, between 20.1% and 28%, between        20.1% and about 27%, between 20.1% and about 26%, between 20.1%        and about 25%, between 20.1% and about 24%, between 21% and 35%,        between 21% and 30%, between 21% and 28%, between 21% and about        26%, or between 21% and about 24%,    -   xiii) the lipid comprises ω6-docosapentaenoic acid        (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xiv) the lipid comprises less than 0.1% of ω6-docosapentaenoic        acid (22:5^(Δ4,7,10,13,16)) in its fatty acid content,    -   xv) the lipid comprises less than 0.1% of one or more or all of        SDA, EPA and ETA in its fatty acid content,    -   xvi) the level of total saturated fatty acids in the total fatty        acid content of the extracted lipid is between about 4% and        about 25%, between about 4% and about 20%, between about 6% and        about 20%, or between about 6% and about 12%,    -   xvii) the level of total monounsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        4% and about 40%, between about 4% and about 35%, between about        8% and about 25%, between 8% and about 22%, between about 15%        and about 40% or between about 15% and about 35%,    -   xviii) the level of total polyunsaturated fatty acids in the        total fatty acid content of the extracted lipid is between about        20% and about 75%, between 30% and 75%, between about 50% and        about 75%, about 60%, about 65%, about 70%, about 75%, or        between about 60% and about 75%,    -   xix) the level of total ω6 fatty acids in the total fatty acid        content of the extracted lipid is between about 35% and about        50%, between about 20% and about 35%, between about 6% and 20%,        less than 20%, less than about 16%, less than about 10%, between        about 1% and about 16%, between about 2% and about 10%, or        between about 4% and about 10%,    -   xx) the level of new ω6 fatty acids in the total fatty acid        content of the extracted lipid is less than about 10%, less than        about 8%, less than about 6%, less than 4%, between about 1% and        about 20%, between about 1% and about 10%, between 0.5% and        about 8%, or between 0.5% and 4%,    -   xxi) the level of total ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 36% and about 65%,        between 36% and about 70%, between 40% and about 60%, between        about 30% and about 60%, between about 35% and about 60%,        between 40% and about 65%, between about 30% and about 65%,        between about 35% and about 65%, about 35%, about 40%, about        45%, about 50%, about 55%, about 60%, about 65% or about 70%,    -   xxii) the level of new ω3 fatty acids in the total fatty acid        content of the extracted lipid is between 21% and about 45%,        between 21% and about 35%, between about 23% and about 35%,        between about 25% and about 35%, between about 27% and about        35%, about 23%, about 25%, about 27%, about 30%, about 35%,        about 40% or about 45%,    -   xxiii) the ratio of total ω6 fatty acids: total ω3 fatty acids        in the fatty acid content of the extracted lipid is between        about 1.0 and about 3.0, between about 0.1 and about 1, between        about 0.1 and about 0.5, less than about 0.50, less than about        0.40, less than about 0.30, less than about 0.20, less than        about 0.15, about 1.0, about 0.1, about 0.10 to about 0.4, or        about 0.2,    -   xxiv) the ratio of new ω6 fatty acids: new ω3 fatty acids in the        fatty acid content of the extracted lipid is between about 1.0        and about 3.0, between about 0.02 and about 0.1, between about        0.1 and about 1, between about 0.1 and about 0.5, less than        about 0.50, less than about 0.40, less than about 0.30, less        than about 0.20, less than about 0.15, about 0.02, about 0.05,        about 0.1, about 0.2 or about 1.0,    -   xxv) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to LA by Δ12-desaturase        of at least about 60%, at least about 70%, at least about 80%,        between about 60% and about 98%, between about 70% and about        95%, or between about 75% and about 90%,    -   xxvi) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to SDA by Δ6-desaturase of at        least about 30%, at least about 40%, at least about 50%, at        least about 60%, at least about 70%, between about 30% and about        70%, between about 35% and about 60%, or between about 50% and        about 70%,    -   xxvii) the fatty acid composition of the lipid is based on an        efficiency of conversion of SDA to ETA acid by Δ6-elongase of at        least about 60%, at least about 70%, at least about 75%, between        about 60% and about 95%, between about 70% and about 88%, or        between about 75% and about 85%,    -   xxviii) the fatty acid composition of the lipid is based on an        efficiency of conversion of ETA to EPA by Δ5-desaturase of at        least about 60%, at least about 70%, at least about 75%, between        about 60% and about 99%, between about 70% and about 99%, or        between about 75% and about 98%,    -   xxix) the fatty acid composition of the lipid is based on an        efficiency of conversion of EPA to DPA by Δ5-elongase of at        least about 80%, at least about 85%, at least about 90%, between        about 50% and about 99%, between about 85% and about 99%,        between about 50% and about 95%, or between about 85% and about        95%,    -   xxx) if the level of DHA in the total fatty acid content of the        extracted lipid is between 20.1% and 30% or between 20.1% and        35%, the fatty acid composition of the lipid is based on an        efficiency of conversion of DPA to DHA by &4-desaturase of at        least about 80%, at least about 90%, at least about 93%, between        about 50% and about 95%, between about 80% and about 95%, or        between about 85% and about 95%,    -   xxxi) the fatty acid composition of the lipid is based on an        efficiency of conversion of oleic acid to DHA and/or DPA of at        least about 10%, at least about 15%, at least about 20%, at        least about 25%, about 20%, about 25%, about 30%, between about        10% and about 50%, between about 10% and about 30%, between        about 10% and about 25% or between about 20% and about 30%,    -   xxxii) the fatty acid composition of the lipid is based on an        efficiency of conversion of LA to DHA and/or DPA of at least        about 15%, at least about 20%, at least about 22%, at least        about 25%, at least about 30%, at least about 40%, about 25%,        about 30%, about 35%, about 40%, about 45%, about 50%, between        about 15% and about 50%, between about 20% and about 40%, or        between about 20% and about 30%,    -   xxxiii) the fatty acid composition of the lipid is based on an        efficiency of conversion of ALA to DHA and/or DPA of at least        about 17%, at least about 22%, at least about 24%, at least        about 30%, about 30%, about 35%, about 40%, about 45%, about        50%, about 55%, about 60%, between about 22% and about 70%,        between about 17% and about 55%, between about 22% and about        40%, or between about 24% and about 40%,    -   xxxiv) the total fatty acid in the extracted lipid has less than        1.5% C20:1, less than 1% C20:1 or about 1% C20:1,    -   xxxv) the triacylglycerol (TAG) content of the lipid is at least        about 70%, at least about 80%, at least about 90%, at least 95%,        between about 70% and about 99%, or between about 90% and about        99%,    -   xxxvi) the lipid comprises diacylglycerol (DAG), which DAG        preferably comprises DHA and/or DPA,    -   xxxvii) the lipid comprises less than about 10%, less than about        5%, less than about 1%, or between about 0.001% and about 5%,        free (non-esterified) fatty acids and/or phospholipid, or is        essentially free thereof,    -   xxxviii) at least 70%, at least 72% or at least 80%, of the DHA        and/or DPA esterified in the form of TAG is in the sn-1 or sn-3        position of the TAG,    -   xxxix) the most abundant DHA-containing TAG species in the lipid        is DHA/18:3/18:3 (TAG 58:12), the lipid comprises tri-DHA TAG        (TAG 66:18), and    -   xl) the level of DPA in the total fatty acid content of the        extracted lipid is about 7%, about 8%, about 9%, about 10%,        about 12%, about 15%, about 18%, about 20%, about 22%, about        24%, about 26%, about 28%, about 31%, between about 7% and about        31%, between about 7% and about 28%, between about 10% and 35%,        between about 10% and about 30%, between about 10% and about        25%, between about 10% and about 22%, between about 14% and 35%,        between about 16% and 35%, between about 16% and about 30%,        between about 16% and about 25%, or between about 16% and about        22%, optionally wherein the level of DHA is less than 0.5% of        the total fatty acid content of the extracted lipid.

In another embodiment, the extracted lipid has one or more of thefollowing features

-   -   i) the level of palmitic acid in the total fatty acid content of        the extracted plant lipid is between 2% and 15%,    -   ii) the level of myristic acid (C14:0) in the total fatty acid        content of the extracted plant lipid is about 0.1%,    -   iii) the level of oleic acid in the total fatty acid content of        the extracted plant lipid is between 1% and 30%,    -   iv) the level of linoleic acid (LA) in the total fatty acid        content of the extracted plant lipid is between 4% and 20%,    -   v) the level of α-linolenic acid (ALA) in the total fatty acid        content of the extracted plant lipid is between 4% and 40%,    -   vi) the level of γ-linolenic acid (GLA) in the total fatty acid        content of the extracted plant lipid is between 0.05% and 7%,    -   vii) the level of stearidonic acid (SDA) in the total fatty acid        content of the extracted plant lipid is between 0.05% and 10%,    -   viii) the level of eicosatetraenoic acid (ETA) in the total        fatty acid content of the extracted plant lipid is less than 6%,    -   ix) the level of eicosatrienoic acid (ETrA) in the total fatty        acid content of the extracted plant lipid is less than 4%,    -   x) the extracted plant lipid comprises less than 0.1% of        ω6-docosapentaenoic acid (22:5^(Δ4,7,10,13,16)) in its fatty        acid content,    -   xi) the level of new ω6 fatty acids in the total fatty acid        content of the extracted plant lipid is less than 10%,    -   xii) the ratio of total ω6 fatty acids: total ω3 fatty acids in        the fatty acid content of the extracted plant lipid is between        1.0 and 3.0, or between 0.1 and 1,    -   xiii) the ratio of new ω6 fatty acids: new ω3 fatty acids in the        fatty acid content of the extracted plant lipid is between 1.0        and 3.0, between 0.02 and 0.1, or between 0.1 and 1,    -   xiv) the fatty acid composition of the extracted plant lipid is        based on an efficiency of conversion of oleic acid to DHA of at        least 10%,    -   xv) the fatty acid composition of the extracted plant lipid is        based on an efficiency of conversion of LA to DHA of at least        15%,    -   xvi) the fatty acid composition of the extracted plant lipid is        based on an efficiency of conversion of ALA to DHA of at least        17%,    -   xvii) the total fatty acid in the extracted plant lipid has less        than 1.5% C20:1, and    -   xviii) the triacylglycerol (TAG) content of the extracted plant        lipid is at least 70%, and may be characterised by one or more        of the following features    -   xix) the extracted plant lipid comprises diacylglycerol (DAG)        which comprises DHA,    -   xx) the extracted plant lipid comprises less than 10% free        (non-esterified) fatty acids and/or phospholipid, or is        essentially free thereof,    -   xxi) at least 70% of the DHA esterified in the form of TAG is in        the sn-1 or sn-3 position of the TAG,    -   xxii) the most abundant DHA-containing TAG species in the        extracted plant lipid is DHA/18:3/18:3 (TAG 58:12), and    -   xxiii) the extracted plant lipid comprises tri-DHA TAG (TAG        66:18).

In an embodiment, the level of eicosapentaenoic acid (EPA) in the totalfatty acid content of the extracted plant lipid is between 0.05% and10%.

In another embodiment, where DHA is present between 20.1% and 35%, thelevel of docosapentaenoic acid (DPA) in the total fatty acid content ofthe extracted plant lipid is less than about 4%.

In a further embodiment, the level of DHA in the total fatty acidcontent of the extracted plant lipid is between 20.1% and 30%.

In another embodiment, the extracted lipid is in the form of an oil,wherein at least about 90%, least about 95%, at least about 98%, orbetween about 95% and about 98%, by weight of the oil is the lipid.

In a preferred embodiment of the first two aspects above, the lipid oroil, preferably a seedoil, has the following features: in the totalfatty acid content of the lipid or oil, the level of DHA is betweenabout 20.1% and 30% or between 20.1% and 35%, the level of palmitic acidis between about 2% and about 16%, the level of myristic acid is lessthan about 6%, the level of oleic acid is between about 1% and about30%, the level of LA is between about 4% and about 35%, ALA is present,the level of total saturated fatty acids in the total fatty acid contentof the extracted lipid is between about 4% and about 25%, the ratio oftotal ω6 fatty acids: total ω3 fatty acids in the fatty acid content ofthe extracted lipid is between 0.05 and about 3.0, and thetriacylglycerol (TAG) content of the lipid is at least about 70%, andoptionally the lipid is essentially free of cholesterol and/or the lipidcomprises tri-DHA TAG (TAG 66:18). More preferably, the lipid or oil,preferably a seedoil, additionally has one or more or all of thefollowing features: at least 70% of the DHA is esterified at the sn-1 orsn-3 position of triacylglycerol (TAG), ALA is present at a level ofbetween 4% and 40% of the total fatty acid content, GLA is presentand/or the level of GLA is less than 4% of the total fatty acid content,the level of SDA is between 0.05% and about 10%, the level of ETA isless than about 4%, the level of EPA is between 0.05% and about 10%, thelevel of DPA is between 0.05% and about 8%, the level of totalmonounsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 4% and about 35%, the level of totalpolyunsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 20% and about 75%, the ratio of new ω6fatty acids: new ω3 tatty acids in the fatty acid content of theextracted lipid is between about 0.03 and about 3.0, preferably lessthan about 0.50, the fatty acid composition of the lipid is based on: anefficiency of conversion of oleic acid to LA by Δ12-desaturase of atleast about 60%, an efficiency of conversion of SDA to ETA acid byΔ6-elongase of at least about 60%, an efficiency of conversion of EPA toDPA by Δ5-elongase of between about 50% and about 95%, an efficiency ofconversion of DPA to DHA by Δ4-desaturase of between about 50% and about95%, an efficiency of conversion of oleic acid to DHA of at least about10%. Most preferably, at least 81% of the DHA is esterified at the sn-1or sn-3 position of triacylglycerol (TAG).

In a preferred embodiment of the third aspect above, the lipid or oil,preferably a seedoil, has the following features: in the total fattyacid content of the lipid or oil, the level of DPA is between about 7%and 30% or between about 7% and 35%, the level of palmitic acid isbetween about 2% and about 16%, the level of myristic acid is less than1%, the level of oleic acid is between about 1% and about 30%, the levelof LA is between about 4% and about 35%, ALA is present, the level oftotal saturated fatty acids in the total fatty acid content of theextracted lipid is between about 4% and about 25%, the ratio of total ω6fatty acids: total ω3 fatty acids in the fatty acid content of theextracted lipid is between 0.05 and about 3.0, and the triacylglycerol(TAG) content of the lipid is at least about 70%, and optionally thelipid is essentially free of cholesterol and/or the lipid comprisestri-DPA TAG (TAG 66:15). More preferably, the lipid or oil, preferably aseedoil, additionally has one or more or all of the following features:at least 70% of the DPA is esterified at the sn-1 or sn-3 position oftriacylglycerol (TAG), ALA is present at a level of between 4% and 40%of the total fatty acid content, GLA is present and/or the level of GLAis less than 4% of the total fatty acid content, the level of SDA isbetween 0.05% and about 10%, the level of ETA is less than about 4%, thelevel of EPA is between 0.05% and about 10%, the level of totalmonounsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 4% and about 35%, the level of totalpolyunsaturated fatty acids in the total fatty acid content of theextracted lipid is between about 20% and about 75%, the ratio of new ω6fatty acids: new ω3 fatty acids in the fatty acid content of theextracted lipid is between about 0.03 and about 3.0, preferably lessthan about 0.50, the fatty acid composition of the lipid is based on: anefficiency of conversion of oleic acid to LA by Δ12-desaturase of atleast about 60%, an efficiency of conversion of SDA to ETA acid byΔ6-elongase of at least about 60%, an efficiency of conversion of EPA toDPA by Δ5-elongase of between about 50% and about 95%, an efficiency ofconversion of oleic acid to DPA of at least about 10%. Most preferably,at least 81% of the DPA is esterified at the sn-1 or sn-3 position oftriacylglycerol (TAG).

In another preferred embodiment of the fourth aspect above, the lipid oroil, preferably a seedoil, comprising DHA and/or DPA has the followingfeatures: in the total fatty acid content of the lipid or oil, the levelof palmitic acid is between about 2% and about 16%, the level ofmyristic acid is less than 1%, the level of oleic acid is between about1% and about 30%, the level of LA is between about 4% and about 35%, ALAis present, the level of total saturated fatty acids in the total fattyacid content of the extracted lipid is between about 4% and about 25%,the ratio of total ω6 fatty acids: total ω3 fatty acids in the fattyacid content of the extracted lipid is between 0.05 and about 3.0, thetriacylglycerol (TAG) content of the lipid is at least about 70%, andoptionally the lipid comprises tri-DHA TAG (TAG 66:18) and/or tri-DPATAG (TAG 66:15), wherein at least 35% of the DPA and/or DHA esterifiedin the form of triacylglycerol (TAG) is esterified at the sn-2 positionof the TAG. More preferably, the lipid or oil, preferably a seedoil,additionally has one or more or all of the following features: ALA ispresent at a level of between 4% and 40% of the total fatty acidcontent, GLA is present and/or the level of GLA is less than 4% of thetotal fatty acid content, the level of SDA is between 0.05% and about10%, the level of ETA is less than about 4%, the level of EPA is between0.05% and about 10%, the level of total monounsaturated fatty acids inthe total fatty acid content of the extracted lipid is between about 4%and about 35%, the level of total polyunsaturated fatty acids in thetotal fatty acid content of the extracted lipid is between about 20% andabout 75%, the ratio of new ω6 fatty acids: new ω3 fatty acids in thefatty acid content of the extracted lipid is between about 0.03 andabout 3.0, preferably less than about 0.50, the fatty acid compositionof the lipid is based on: an efficiency of conversion of oleic acid toLA by Δ12-desaturase of at least about 60%, an efficiency of conversionof SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiencyof conversion of EPA to DPA by Δ5-elongase of between about 50% andabout 95%, an efficiency of conversion of DPA to DHA by Δ4-desaturase(if present) of between about 50% and about 95%, an efficiency ofconversion of oleic acid to DPA of at least about 10%.

In the context of the extracted lipid or oil of the invention, the levelof DHA and/or DPA in the extracted lipid or oil has not been increased,or is substantially the same as, the level of DHA and/or DPA in thelipid or oil of the plant part or microbe prior to extraction. In otherwords, no procedure has been performed to increase the level of DHAand/or DPA in the lipid or oil relative to other fatty acidspost-extraction. As would be apparent, the lipid or oil may subsequentlybe treated by fractionation or other procedures to alter the fatty acidcomposition.

In a further preferred embodiment, the lipid or oil, preferably aseedoil, has the following features: in the total fatty acid content ofthe lipid or oil, the level of DHA is between about 20.1% and 30% orbetween about 20.1% and 35%, the level of palmitic acid is between about2% and about 16%, the level of myristic acid is less than about 6% andpreferably less than 1%, the level of oleic acid is between about 1% andabout 30%, the level of LA is between about 4% and about 35%, ALA ispresent, GLA is present, the level of SDA is between about 0.05% andabout 10%, the level of ETA is less than about 6%, the level of EPA isbetween about 0.05% and about 10%, the level of DPA is between about0.05% and about 8%.

In another preferred embodiment, the lipid or oil, preferably a seedoiland more preferably a Brassica seedoil such as mustard oil or canolaoil, has the following features: in the total fatty acid content of thelipid or oil, the level of DPA is between about 7% and 35%, the level ofpalmitic acid is between about 2% and about 16%, the level of myristicacid is less than about 6% and preferably less than 1%, the level ofoleic acid is between about 1% and about 30%, the level of LA is betweenabout 4% and about 35%, ALA is present, the level of SDA is betweenabout 0.05% and about 10%, the level of ETA is less than about 6%, thelevel of EPA is between about 0.05% and about 10%. DHA may or may not bepresent in the lipid or oil. Preferably, DHA, if present, is present ata level of not more than 0.5% of the total fatty acid content of thelipid or oil and more preferably is absent from the total fatty acidcontent of the lipid or oil. Optionally, the lipid is essentially freeof cholesterol and/or the lipid comprises tri-DPA TAG (TAG 66:15). Morepreferably, the lipid or oil, preferably a seedoil, additionally has oneor more or all of the following features: at least 70% of the DPA isesterified at the sn-1 or sn-3 position of triacylglycerol (TAG), ALA ispresent at a level of between 4% and 40% of the total fatty acidcontent, GLA is present and/or the level of GLA is less than 4% of thetotal fatty acid content, the level of SDA is between 0.05% and about10%, the level of ETA is less than about 4%, the level of EPA is between0.05% and about 10%, the level of total monounsaturated fatty acids inthe total fatty acid content of the extracted lipid is between about 4%and about 35%, the level of total polyunsaturated fatty acids in thetotal fatty acid content of the extracted lipid is between about 20% andabout 75%, the ratio of new ω6 fatty acids: new ω3 fatty acids in thefatty acid content of the extracted lipid is between about 0.03 andabout 3.0, preferably less than about 0.50, the fatty acid compositionof the lipid is based on: an efficiency of conversion of oleic acid toLA by Δ12-desaturase of at least about 60%, an efficiency of conversionof SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiencyof conversion of EPA to DPA by Δ5-elongase of between about 50% andabout 95%, an efficiency of conversion of oleic acid to DPA of at leastabout 10%. In an embodiment, at least 81% of the DPA is esterified atthe sn-1 or sn-3 position of triacylglycerol (TAG). Alternatively, atleast 35% of the DPA esterified in the form of TAG is esterified at thesn-2 position of TAG.

In a further embodiment, the extracted lipid of the invention furthercomprises one or more sterols, preferably plant sterols.

In another embodiment, the extracted lipid is in the form of an oil, andcomprises less than about 10 mg of sterols/g of oil, less than about 7mg of sterols/g of oil, between about 1.5 mg and about 10 mg ofsterols/g of oil, or between about 1.5 mg and about 7 mg of sterols/g ofoil.

Examples of sterols which can be in the extracted lipid include, but arenot necessarily limited to, one or more or all ofcampesterol/24-methylcholesterol, Δ5-stigmasterol, eburicol,β-sitosterol/24-ethylcholesterol, Δ5-avenasterol/isofucosterol,Δ7-stigmasterol/stigmast-7-en-3β-ol, and Δ7-avenasterol.

In an embodiment, the plant species is one listed in Table 14, such ascanola, and the level of sterols are about the same as that listed inTable 14 for that particular plant species. The plant species may bemustard (B. juncea) or C. sativa and comprise a level of sterols aboutthat found in wild-type mustard or C. sativa extracted oil,respectively.

In an embodiment, the extracted plant lipid comprises one or more or allof campesterol/24-methylcholesterol, Δ5-stigmasterol, eburicol,β-sitosterol/24-ethylcholesterol, Δ5-avenasterol/isofucosterol,Δ7-stigmasterol/stigmast-7-en-3β-ol, and Δ7-avenasterol, or which has asterol content essentially the same as wild-type canola oil.

In an embodiment, the extracted lipid has a sterol content essentiallythe same as wild-type canola oil, mustard oil or C. sativa oil.

In an embodiment, the extracted lipid comprises less than about 0.5 mgof cholesterol/g of oil, less than about 0.25 mg of cholesterol/g ofoil, between about 0 mg and about 0.5 mg of cholesterol/g of oil, orbetween about 0 mg and about 0.25 mg of cholesterol/g of oil, or whichis essentially free of cholesterol.

In a further embodiment, the lipid is an oil, preferably oil from anoilseed. Examples of such oils include, but are not limited to, Brassicasp. oil such as for example canola oil or mustard oil, Gossypiumhirsutum oil, Linum usitatissimum oil, Helianthus sp. oil, Carthamustinctorius oil, Glycine max oil, Zea mays oil, Arabidopsis thaliana oil,Sorghum bicolor oil, Sorghum vulgare oil, Avena sativa oil, Trifoliumsp. oil, Elaesis guineenis oil, Nicotiana benthamiana oil, Hordeumvulgare oil, Lupinus angustifolius oil, Oryza sativa oil, Oryzaglaberrima oil, Camelina sativa oil, Crambe abyssinica oil,Miscanthus×giganteus oil, or Miscanthus sinensis oil. More preferably,the oil is a Brassica sp. oil, a Camelina sativa oil or a Glycine max(soybean) oil. In an embodiment the lipid comprises or is Brassica sp.oil such as Brassica napus oil or Brassica juncea oil, Gossypiumhirsutum oil, Linum usitatissimum oil, Helianthus sp. oil, Carthamustinctorius oil, Glycine max oil, Zea mays oil, Elaesis guineenis oil,Nicotiana benthamiana oil, Lupinus angustifolius oil, Camelina sativaoil, Crambe abyssinica oil, Miscanthus×giganteus oil, or Miscanthussinensis oil. In a further embodiment, the oil is canola oil, mustard(B. juncea) oil, soybean (Glycine max) oil, Camelina sativa oil orArabidopsis thaliana oil. In an alternate embodiment, the oil is a plantoil other than A. thaliana oil and/or other than C. sativa oil. In anembodiment, the plant oil is an oil other than G. max (soybean) oil. Inan embodiment, the oil was obtained from a plant grown under standardconditions, for Example as described in Example 1, or from a plant grownin the field or in a glasshouse under standard conditions.

In another aspect, the present invention provides a process forproducing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA) andγ-linolenic acid (GLA), ω3 fatty acids which comprise α-linolenic acid(ALA), stearidonic acid (SDA), docosapentaenoic acid (DPA) anddocosahexaenoic acid (DHA), and optionally one or more ofeicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein thelevel of DHA in the total fatty acid content of extractable lipid in theplant part is between 20.1% and 30% or between 20.1% and 35%, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of theextracted lipid is between 20.1% and 30% or between 20.1% and 35%.

In a further aspect, the present invention provides a process forproducing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA) andγ-linolenic acid (GLA), ω3 fatty acids which comprise α-linolenic acid(ALA), stearidonic acid (SDA), docosapentaenoic acid (DPA) anddocosahexaenoic acid (DHA), and optionally one or more ofeicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein thelevel of palmitic acid in the total fatty acid content of the extractedlipid is between about 2% and 16%, and wherein the level of myristicacid (C14:0) in the total fatty acid content of the extracted lipid, ifpresent, is less than 1%, and wherein the level of DHA in the totalfatty acid content of extractable lipid in the plant part is between20.1% and 30% or between 20.1% and 35%, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of theextracted lipid is between 20.1% and 30% or between 20.1% and 35%.

In a further aspect, the present invention provides a process forproducing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA), stearidonic acid(SDA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), andoptionally one or more of eicosapentaenoic acid (EPA) andeicosatetraenoic acid (ETA), wherein the level of palmitic acid in thetotal fatty acid content of the extracted lipid is between about 2% and16%, and wherein the level of myristic acid (C14:0) in the total fattyacid content of the extracted lipid, if present, is less than 1%, andwherein the level of DHA in the total fatty acid content of extractablelipid in the plant part is between 20.1% and 30% or between 20.1% and35%, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of theextracted lipid is between 20.1% and 30% or between 20.1% and 35%.

In an embodiment of the three above aspect, the invention provides aprocess for producing extracted plant lipid, comprising the steps of

i) obtaining a plant part comprising lipid, the lipid comprising fattyacids in an esterified form, wherein the lipid has a fatty acidcomposition comprising oleic acid, palmitic acid, ω6 fatty acids whichcomprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenicacid (ALA) and docosahexaenoic acid (DHA), and one or more ofstearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoicacid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHAin the total fatty acid content of the extracted lipid is between 20.1%and 30% or between 20.1% and 35%, preferably between 30% and 35%, (ii)the level of palmitic acid in the total fatty acid content of theextracted lipid is between 2% and 16%, (iii) the level of myristic acid(C14:0) in the total fatty acid content of the extracted lipid is lessthan 1%, (iv) the level of oleic acid in the total fatty acid content ofthe extracted lipid is between 1% and 30%, (v) the level of linoleicacid (LA) in the total fatty acid content of the extracted lipid isbetween 4% and 35%, (vi) the level of α-linolenic acid (ALA) in thetotal fatty acid content of the extracted lipid is between 4% and 40%,(vii) the level of eicosatrienoic acid (ETrA) in the total fatty acidcontent of the extracted lipid is less than 4%, (viii) the level oftotal saturated fatty acids in the total fatty acid content of theextracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fattyacids: total ω3 fatty acids in the fatty acid content of the extractedlipid is between 0.05 and 1, (x) the triacylglycerol (TAG) content ofthe lipid is at least 70%, and (xi) at least 70% of the DHA esterifiedin the form of TAG is in the sn-1 or sn-3 position of the TAG, and

ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of theextracted lipid is between about 20.1% and 30% or between 20.1% and 35%,preferably between 30% and 35%. Preferably, at least 81% or at least 90%of the DHA esterified in the form of TAG is in the sn-1 or sn-3 positionof the TAG.

In a further aspect, the invention provides a process for producingextracted plant lipid or microbial lipid, comprising the steps of

i) obtaining a plant part or microbial cells comprising lipid, the lipidcomprising fatty acids in an esterified form, the fatty acids comprisingoleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid(LA), ω3 fatty acids which comprise α-linolenic acid (ALA) anddocosapentaenoic acid (DPA), and optionally one or more of stearidonicacid (SDA), eicosapentaenoic acid (EPA), and eicosatetraenoic acid(ETA), wherein the level of DPA in the total fatty acid content of thelipid of the plant part or microbial cells between about 7% and 35%, and

ii) extracting lipid from the plant part or microbial cells,

wherein the level of DPA in the total fatty acid content of theextracted lipid is between about 7% and 35%. In an embodiment, the levelof DPA in the total fatty acid content of the extracted lipid is betweenabout 7% and 20%, or between 20.1% and 35%.

In an embodiment of the above aspect, the invention provides a processfor producing extracted plant lipid or microbial lipid, comprising thesteps of

i) obtaining a plant part or microbial cells comprising lipid, the lipidcomprising fatty acids in an esterified form, wherein the lipid has afatty acid composition comprising oleic acid, palmitic acid, ω6 fattyacids which comprise linoleic acid (LA), ω3 fatty acids which compriseα-linolenic acid (ALA) and docosahexaenoic acid (DPA), and one or moreof stearidonic acid (SDA), eicosapentaenoic acid (EPA), andeicosatetraenoic acid (ETA), wherein (i) the level of DPA in the totalfatty acid content of the extracted lipid is between 7% and 30% orbetween 7% and 35%, preferably between 30% and 35%, (ii) the level ofpalmitic acid in the total fatty acid content of the extracted lipid isbetween 2% and 16%, (iii) the level of myristic acid (C14:0) in thetotal fatty acid content of the extracted lipid is less than 6%,preferably less than 1%, (iv) the level of oleic acid in the total fattyacid content of the extracted lipid is between 1% and 30%, (v) the levelof linoleic acid (LA) in the total fatty acid content of the extractedlipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) inthe total fatty acid content of the extracted lipid is between 4% and40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fattyacid content of the extracted lipid is less than 4%, (viii) the level oftotal saturated fatty acids in the total fatty acid content of theextracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fattyacids: total ω3 fatty acids in the fatty acid content of the extractedlipid is between 0.05 and 1, (x) the triacylglycerol (TAG) content ofthe lipid is at least 70%, and (xi) at least 70% of the DPA esterifiedin the form of TAG is in the sn-1 or sn-3 position of the TAG and

ii) extracting lipid from the plant part,

wherein the level of DPA in the total fatty acid content of theextracted lipid is between about 7% and 30% or between 7% and 35%,preferably between 30% and 35%. Preferably, at least 81% or at least 90%of the DPA esterified in the form of TAG is in the sn-1 or sn-3 positionof the TAG.

In another aspect, the present invention provides a process forproducing extracted lipid, comprising the steps of

i) obtaining cells, preferably a plant part comprising the cells ormicrobial cells, comprising lipid, the lipid comprising fatty acids inan esterified form, the fatty acids comprising docosapentaenoic acid(DPA) and/or docosahexaenoic acid (DHA), wherein at least 35% of the DPAand/or DHA esterified in the form of triacylglycerol (TAG) is esterifiedat the sn-2 position of the TAG, and

ii) extracting lipid from the cells,

wherein at least 35% of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) in the total fatty acid content of the extractedlipid is esterified at the sn-2 position of the TAG. In an embodiment,the extracted lipid produced by the process is further characterised byone or more or all of (i) it comprises fatty acids comprising oleicacid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA),ω3 fatty acids which comprise α-linolenic acid (ALA) and optionally oneor more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), andeicosatetraenoic acid (ETA), (ii) at least about 40%, at least about45%, at least about 48%, between 35% and about 60%, or between 35% andabout 50%, of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) is esterified at the sn-2 position of the TAG, and(iii) the level of DPA and/DHA in the total fatty acid content of theextracted lipid is between about 1% and 35%, or between about 7% and 35%or between about 20.1% and 35%. In embodiments of this aspect, the levelof DPA and/or DHA in the total fatty acid content of the extracted lipidis about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about18%, about 20%, about 22%, about 24%, about 26%, about 28%, about 30%,between about 7% and about 28%, between about 7% and about 25%, betweenabout 10% and 35%, between about 10% and about 30%, between about 10%and about 25%, between about 10% and about 22%, between about 14% and35%, between about 16% and 35%, between about 16% and about 30%, betweenabout 16% and about 25%, or between about 16% and about 22%. Inpreferred embodiments, the extracted lipid is characterised by (i) and(ii), (i) and (iii) or (ii) and (iii), more preferably all of (i), (ii)and (iii). Preferably, the extracted lipid is further characterised by alevel of palmitic acid in the total fatty acid content of the extractedlipid which is between about 2% and 16%, and a level of myristic acid(C14:0) in the total fatty acid content of the extracted lipid, ifpresent, is less than 1%.

In an embodiment of the above aspect, the invention provides a processfor producing extracted lipid, comprising the steps of

i) obtaining cells, preferably a plant part comprising the cells ormicrobial cells, comprising lipid, the lipid comprising fatty acids inan esterified form, the fatty acids comprising docosapentaenoic acid(DPA) and/or docosahexaenoic acid (DHA), and further comprising oleicacid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA),ω3 fatty acids which comprise α-linolenic acid (ALA), and one or more ofstearidonic acid (SDA), eicosapentaenoic acid (EPA), andeicosatetraenoic acid (ETA), wherein (i) the level of palmitic acid inthe total fatty acid content of the extracted lipid is between 2% and16%, (ii) the level of myristic acid (C14:0) in the total fatty acidcontent of the extracted lipid is less than 1%, (iii) the level of oleicacid in the total fatty acid content of the extracted lipid is between1% and 30%, (iv) the level of linoleic acid (LA) in the total fatty acidcontent of the extracted lipid is between 4% and 35%, (v) the level ofα-linolenic acid (ALA) in the total fatty acid content of the extractedlipid is between 4% and 40%, (vi) the level of eicosatrienoic acid(ETrA) in the total fatty acid content of the extracted lipid is lessthan 4%, (vii) the level of total saturated fatty acids in the totalfatty acid content of the extracted lipid is between 4% and 25%, (viii)the ratio of total ω6 fatty acids: total ω3 fatty acids in the fattyacid content of the extracted lipid is between 0.05 and 1, (ix) thetriacylglycerol (TAG) content of the lipid is at least 70%, and (x) atleast 35% of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) is esterified at the sn-2 position of the TAG, and

ii) extracting lipid from the plant part,

wherein at least 35% of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) in the total fatty acid content of the extractedlipid is esterified at the sn-2 position of the TAG.

The step of obtaining the plant part or microbial cells may compriseharvesting plant parts, preferably seed, from plants that produce theplant parts, recovery of the microbial cells from cultures of suchcells, or obtaining the plant parts or microbial cells by purchase froma producer or supplier, or by importation. The process may comprise astep of determining the fatty acid composition of the lipid in a sampleof the plant parts or microbial cells, or of the extracted lipid.

In a preferred embodiment, the extracted lipid obtained by a process ofthe invention has, where relevant, one or more of the features definedherein, for example as defined above in relation to the first fouraspects.

Embodiments of each of the five above aspects are described in furtherdetail below. As the skilled person would understand, any embodimentsdescribed which are broader than the corresponding feature in an aboveaspect do not apply to that aspect.

In an embodiment, the plant part is a seed, preferably an oilseed.Examples of such seeds include, but are not limited to, Brassica sp.,Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamustinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghumbicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesisguineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinusangustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, orCrambe abyssinica, preferably a Brassica sp. seed, a C. sativa seed or aG. max (soybean) seed, more preferably a Brassica napus, B. juncea or C.sativa seed. In an embodiment, the plant part is a seed, preferably anoilseed such as Brassica sp. such as Brassica napus or Brassica juncea,Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamustinctorius, Glycine max, Zea mays, Elaesis guineenis, Nicotianabenthamiana, Lupinus angustifolius, Camelina sativa, or Crambeabyssinica, preferably a Brassica napus, B juncea or C. sativa seed. Inan embodiment, the seed is canola seed, mustard seed, soybean seed,Camelina sativa seed or Arabidopsis thaliana seed. In an alternateembodiment, the seed is a seed other than A. thaliana seed and/or otherthan C. sativa seed. In an embodiment, the seed is a seed other thansoybean seed. In an embodiment, the plant part is Brassica sp. seed. Inan embodiment, the seed was obtained from a plant grown under standardconditions, for Example as described in Example 1, or from a plant grownin the field or in a glasshouse under standard conditions.

In another embodiment, the seed comprises at least about 18 mg, at leastabout 22 mg, at least about 26 mg, between about 18 mg and about 100 mg,between about 22 mg and about 70 mg, about 80 mg, between about 30 mgand about 80 mg, or between about 24 mg and about 50 mg, of DHA and/orDPA per gram of seed.

In a further embodiment, the plant part such as a seed comprisesexogenous polynucleotides encoding one of the following sets of enzymes;

i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ6-elongase and a Δ5-elongase,

iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) a Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and anΔ5-elongase,

v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and an Δ5-elongase,

vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase,a Δ9-elongase and a Δ5-elongase,

vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, aΔ4-desaturase, a Δ9-elongase and an Δ5-elongase,

viii) a Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and anΔ5-elongase, and wherein each polynucleotide is operably linked to oneor more promoters that are capable of directing expression of saidpolynucleotides in a cell of the plant part.

In a further embodiment, the plant part such as a seed or recombinantcells such as microbial cells comprise exogenous polynucleotidesencoding one of the following sets of enzymes;

i) an ω3-desaturase and/or a Δ15-desaturase, a Δ6-desaturase, aΔ5-desaturase, a Δ6-elongase and a Δ5-elongase,

ii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongaseand an Δ5-elongase,

iii) a Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an Δ5-elongase,

iv) an ω3-desaturase and/or a Δ15-desaturase, a Δ8-desaturase, aΔ5-desaturase, a Δ9-elongase and a Δ5-elongase,

v) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase andan Δ5-elongase,

vi) a Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ9-elongase and an Δ5-elongase,

and wherein each polynucleotide is operably linked to one or morepromoters that are capable of directing expression of saidpolynucleotides in a cell of the plant part or the cells.

In an embodiment, if the plant part or cells comprise lipid comprisingfatty acids in an esterified form, the fatty acids comprisingdocosapentaenoic acid (DPA) and/or docosahexaenoic acid (DHA), whereinat least 35% of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) is esterified at the sn-2 position of the TAG, thethe plant part such as a seed or recombinant cells such as microbialcells comprise an exogenous polynucleotide encoding an1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), wherein thepolynucleotide is operably linked to one or more promoters that arecapable of directing expression of the polynucleotide in a cell of theplant part or the cells. In a further embodiment, the cells comprisesexogenous polynucleotides encoding one of the following sets of enzymes;

i) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), anω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

ii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

iii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, anΔ5-elongase and optionally a Δ4-desaturase,

iv) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an Δ5-elongase andoptionally a Δ4-desaturase,

v) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), anω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, anΔ5-elongase and optionally a Δ4-desaturase,

vi) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

vii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, anΔ5-elongase and optionally a Δ4-desaturase,

viii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a β-desaturase and/or a Δ15-desaturase, a Δ8-desaturase,a Δ5-desaturase, a Δ9-elongase, an Δ5-elongase and optionally aΔ4-desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell. Preferably, the LPAAT can use a C22 polyunsaturated fatty acyl-CoAsubstrate such as DHA-CoA and/or DPA-CoA.

In an embodiment, the Δ12-desaturase also has ω3-desaturase and/orΔ15-desaturase activity, i.e. the activities are conferred by a singlepolypeptide. Alternatively, the Δ12-desaturase does not haveω3-desaturase activity and does not have Δ15-desaturase activity i.e.the Δ12-desaturase is a separate polypeptide to the polypeptide havingω3-desaturase activity and/or Δ15-desaturase.

In yet a further embodiment, the plant part such as a seed orrecombinant cells such as microbial cells have one or more or all of thefollowing features:

i) the Δ12-desaturase converts oleic acid to linoleic acid in one ormore cells of the plant part or in the recombinant cells with anefficiency of at least about 60%, at least about 70%, at least about80%, between about 60% and about 95%, between about 70% and about 90%,or between about 75% and about 85%,

ii) the ω3-desaturase converts cob fatty acids to ω3 fatty acids in oneor more cells of the plant part or in the recombinant cells with anefficiency of at least about 65%, at least about 75%, at least about85%, between about 65% and about 95%, between about 75% and about 91%,or between about 80% and about 91%,

iii) the Δ6-desaturase converts ALA to SDA in one or more cells of theplant part or in the recombinant cells with an efficiency of at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, between about 30% and about 70%,between about 35% and about 60%, or between about 50% and about 70%,

iv) the Δ6-desaturase converts linoleic acid to γ-linolenic acid in oneor more cells of the plant part or in the recombinant cells with anefficiency of less than about 5%, less than about 2.5%, less than about1%, between about 0.1% and about 5%, between about 0.5% and about 2.5%,or between about 0.5% and about 1%,

v) the Δ6-elongase converts SDA to ETA in one or more cells of the plantpart or in the recombinant cells with an efficiency of at least about60%, at least about 70%, at least about 75%, between about 60% and about95%, between about 70% and about 80%, or between about 75% and about80%,

vi) the Δ5-desaturase converts ETA to EPA in one or more cells of theplant part or in the recombinant cells with an efficiency of at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 90%, between about 60% and about 95%, between about 70%and about 95%, or between about 75% and about 95%,

vii) the Δ5-elongase converts EPA to DPA in one or more cells of theplant part or in the recombinant cells with an efficiency of at leastabout 80%, at least about 85%, at least about 90%, between about 50% andabout 90%, or between about 85% and about 95%,

viii) the Δ4-desaturase converts DPA to DHA in one or more cells of theplant part or in the recombinant cells with an efficiency of at leastabout 80%, at least about 90%, at least about 93%, between about 50% andabout 95%, between about 80% and about 95%, or between about 85% andabout 95%,

ix) the efficiency of conversion of oleic acid to DHA or DPA in one ormore cells of the plant part or in the recombinant cells is at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,about 20%, about 25%, about 30%, between about 10% and about 50%,between about 10% and about 30%, between about 10% and about 25%, orbetween about 20% and about 30%,

x) the efficiency of conversion of LA to DHA or DPA in one or more cellsof the plant part or in the recombinant cells is at least about 15%, atleast about 20%, at least about 22%, at least about 25%, at least about30%, about 25%, about 30%, about 35%, between about 15% and about 50%,between about 20% and about 40%, or between about 20% and about 30%,

xi) the efficiency of conversion of ALA to DHA or DPA in one or morecells of the plant part or in the recombinant cells is at least about17%, at least about 22%, at least about 24%, at least about 30%, about30%, about 35%, about 40%, between about 17% and about 55%, betweenabout 22% and about 35%, or between about 24% and about 35%,

xii) one or more cells of the plant part or the recombinant cellscomprise at least about 25%, at least about 30%, between about 25% andabout 40%, or between about 27.5% and about 37.5%, more ω3 fatty acidsthan corresponding cells lacking the exogenous polynucleotides,

xiii) the Δ6-desaturase preferentially desaturates α-linolenic acid(ALA) relative to linoleic acid (LA),

xiv) the Δ6-elongase also has Δ9-elongase activity,

xv) the Δ12-desaturase also has Δ15-desaturase activity,

xvi) the Δ6-desaturase also has Δ8-desaturase activity,

xvii) the Δ8-desaturase also has Δ6-desaturase activity or does not haveΔ6-desaturase activity,

xviii) the Δ15-desaturase also has ω3-desaturase activity on GLA,

xix) the ω3-desaturase also has Δ15-desaturase activity on LA,

xx) the ω3-desaturase desaturates both LA and/or GLA,

xxi) the ω3-desaturase preferentially desaturates GLA relative to LA,

xxii) one or more or all of the desaturases, preferably theΔ6-desaturase and/or the Δ5-desaturase, have greater activity on anacyl-CoA substrate than a corresponding acyl-PC substrate,

xxiii) the Δ6-desaturase has greater Δ6-desaturase activity on ALA thanLA as fatty acid substrate,

xxiv) the Δ6-desaturase has greater Δ6-desaturase activity on ALA-CoA asfatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate,

xxv) the Δ6-desaturase has at least about a 2-fold greater Δ6-desaturaseactivity, at least 3-fold greater activity, at least 4-fold greateractivity, or at least 5-fold greater activity, on ALA as a substratecompared to LA,

xxvi) the Δ6-desaturase has greater activity on ALA-CoA as fatty acidsubstrate than on ALA joined to the sn-2 position of PC as fatty acidsubstrate,

xxvii) the Δ6-desaturase has at least about a 5-fold greaterΔ6-desaturase activity or at least 10-fold greater activity, on ALA-CoAas fatty acid substrate than on ALA joined to the sn-2 position of PC asfatty acid substrate,

xxviii) the desaturase is a front-end desaturase, and

xxix) the Δ6-desaturase has no detectable Δ5-desaturase activity on ETA.

In yet a further embodiment, the plant part such as a seed or therecombinant cell such as microbial cells has one or more or all of thefollowing features

i) the Δ12-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:4, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:4,

ii) the ω3-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:6, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:6,

iii) the Δ6-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:9, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:9,

iv) the Δ6-elongase comprises amino acids having a sequence as providedin SEQ ID NO:16, a biologically active fragment thereof such as SEQ IDNO:17, or an amino acid sequence which is at least 50% identical to SEQID NO:16 and/or SEQ ID NO:17,

v) the Δ5-desaturase comprises amino acids having a sequence as providedin SEQ ID NO:20, a biologically active fragment thereof, or an aminoacid sequence which is at least 50% identical to SEQ ID NO:20,

vi) the Δ5-elongase comprises amino acids having a sequence as providedin SEQ ID NO:25, a biologically active fragment thereof, or an aminoacid sequence which is at least 50% identical to SEQ ID NO:25, and

vii) the Δ4-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:28, a biologically active fragment thereof, or anamino acid sequence which is at least 50% identical to SEQ ID NO:28.

In an embodiment, the plant part such as a seed or the recombinant cellssuch as microbial cells further comprise(s) an exogenous polynucleotideencoding a diacylglycerol acyltransferase (DGAT), monoacylglycerolacyltransferase (MGAT), glycerol-3-phosphate acyltransferase (GPAT),1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) preferably an LPAATwhich can use a C22 polyunsaturated fatty acyl-CoA substrate such asDHA-CoA and/or DPA-CoA, acyl-CoA:lysophosphatidylcholine acyltransferase(LPCAT), phospholipase A₂ (PLA₂), phospholipase C (PLC), phospholipase D(PLD), CDP-choline diacylglycerol choline phosphotransferase (CPT),phoshatidylcholine diacylglycerol acyltransferase (PDAT),phosphatidylcholine:diacylglycerol choline phosphotransferase (PDCT),acyl-CoA synthase (ACS), or a combination of two or more thereof.

In another embodiment, the plant part such as a seed or the recombinantcells such as microbial cells further comprise(s) an introduced mutationor an exogenous polynucleotide which down regulates the productionand/or activity of an endogenous enzyme in a cell of the plant partselected from FAE1, DGAT, MGAT, GPAT, LPAAT, LPCAT, PLA₂, PLC, PLD, CPT,PDAT, a thioesterase such as FATB, or a Δ12-desaturase, or a combinationof two or more thereof.

In a further embodiment, at least one, or all, of the promoters are seedspecific promoters. In an embodiment, at least one, or all, of thepromoters have been obtained from an oil biosynthesis or accumulationgene such as a gene encoding oleosin, or from a seed storage proteingenes such as a gene encoding conlinin.

In another embodiment, the promoter(s) directing expression of theexogenous polynucleotides encoding the Δ4-desaturase and the Δ5-elongaseinitiate expression of the polynucleotides in developing seed of theplant or the recombinant cells such as the microbial cells before, orreach peak expression before, the promoter(s) directing expression ofthe exogenous polynucleotides encoding the Δ12-desaturase and theω3-desaturase.

In a further embodiment, the exogenous polynucleotides are covalentlylinked in a DNA molecule, preferably a T-DNA molecule, integrated intothe genome of cells of the plant part or the recombinant cells such asthe microbial cells and preferably where the number of such DNAmolecules integrated into the genome of the cells of the plant part orthe recombinant cells is not more than one, two or three, or is two orthree.

In yet another embodiment, the plant part comprises at least twodifferent, exogenous polynucleotides each encoding a Δ6-desaturase whichhave the same or different amino acid sequences.

In a further embodiment, the total oil content of the plant partcomprising the exogenous polynucleotides is at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, between about 50% andabout 80%, or between about 80% and about 100% of the total oil contentof a corresponding plant part lacking the exogenous polynucleotides. Ina further embodiment, the seed comprising the exogenous polynucleotideshas a seed weight at least about 40%, at least about 50%, at least about60%, at least about 70%, between about 50% and about 80%, or betweenabout 80% and about 100% of the weight of a corresponding seed lackingthe exogenous polynucleotides.

In another embodiment, the lipid is in the form of an oil, preferably aseedoil from an oilseed, and wherein at least about 90%, or about least95%, at least about 98%, or between about 95% and about 98%, by weightof the lipid is triacylglycerols.

In a further embodiment, the process further comprises treating thelipid to increase the level of DHA and/or DPA as a percentage of thetotal fatty acid content. For example, the treatment comprisestransesterification. For example, the lipid such as canola oil may betreated to convert the fatty acids in the oil to alkyl esters such asmethyl or ethyl esters, which may then be fractionated to enrich thelipid or oil for the DHA and/or DPA. In embodiments, the fatty acidcomposition of the lipid after such treatment comprises at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% DHAand/or DPA. The ratio of DHA:DPA in the lipid after treatment ispreferably greater than 2:1, or alternatively less than 0.5:1.Alternatively, the level of DHA in the total fatty acid content of thelipid after treatment is less than 2.0% or less than 0.5%, preferably isabsent from the lipid.

Also provided is lipid, or oil comprising the lipid, produced using aprocess of the invention.

In another aspect, the present invention provides a process forproducing methyl or ethyl esters of polyunsaturated fatty acids, theprocess comprising reacting triacylglycerols in extracted plant lipid,or during the process of extraction, with methanol or ethanol,respectively, wherein the extracted plant lipid comprises fatty acidsesterified in the form of TAG, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoicacid (DHA), and optionally one or more of stearidonic acid (SDA),eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) andeicosatetraenoic acid (ETA), wherein the level of DHA in the total fattyacid content of the extracted lipid is between about 20.1% and 30%, orbetween 20.1% and 35%, preferably between 30% and 35%, thereby producingthe methyl or ethyl esters of polyunsaturated fatty acids.

In another aspect, the present invention provides a process forproducing methyl or ethyl esters of polyunsaturated fatty acids, theprocess comprising reacting triacylglycerols in extracted plant lipid,or during the process of extraction, with methanol or ethanol,respectively, wherein the extracted plant lipid comprises fatty acidsesterified in the form of TAG, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA), and docosapentaenoicacid (DPA), and optionally one or more of stearidonic acid (SDA),eicosapentaenoic acid (EPA), and eicosatetraenoic acid (ETA), whereinthe level of DPA in the total fatty acid content of the extracted lipidis between about 7% and 35%, preferably between 20.1% and 30% or between20.1% and 35%, thereby producing the methyl or ethyl esters ofpolyunsaturated fatty acids.

In another aspect, the present invention provides a process forproducing methyl or ethyl esters of docosapentaenoic acid (DPA) and/ordocosahexaenoic acid (DHA), the process comprising reactingtriacylglycerols (TAG) in extracted plant lipid, or during the processof extraction, with methanol or ethanol, respectively, wherein theextracted plant lipid comprises fatty acids in an esterified form, thefatty acids comprising docosapentaenoic acid (DPA) and/ordocosahexaenoic acid (DHA), wherein at least 35% of the DPA and/or DHAesterified in the form of TAG is esterified at the sn-2 position of theTAG, thereby producing the methyl or ethyl esters of polyunsaturatedfatty acids.

In a preferred embodiment, the lipid which is used in the process of theabove three aspects has one or more of the features defined herein inthe context of the extracted lipid or oil of the invention.

In another aspect, the present invention provides an oilseed plant orpart thereof such as a seed comprising

a) lipid in its seed, the lipid comprising fatty acids in an esterifiedform, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase        and an Δ5-elongase, or    -   ii) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase        and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or moreseed-specific promoters that are capable of directing expression of saidpolynucleotides in developing seed of the plant, wherein the fatty acidscomprise oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA) and γ-linolenic acid (GLA), ω3 fatty acids whichcomprise α-linolenic acid (ALA), stearidonic acid (SDA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), andoptionally eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid(ETA), and wherein the level of DHA in the total fatty acid content ofthe lipid of the seed is between 20.1% and 30%, or between 20.1% and35%, preferably between 30% and 35%.

In another aspect, the present invention provides an oilseed plant orpart thereof such as a seed comprising

a) lipid in its seed, the lipid comprising fatty acids in an esterifiedform, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase        and an Δ5-elongase, or    -   ii) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase        and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or moreseed-specific promoters that are capable of directing expression of saidpolynucleotides in developing seed of the plant, wherein the fatty acidscomprise oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid(ALA), stearidonic acid (SDA), docosapentaenoic acid (DPA) anddocosahexaenoic acid (DHA), and optionally eicosapentaenoic acid (EPA)and/or eicosatetraenoic acid (ETA), and wherein the level of DHA in thetotal fatty acid content of the lipid of the seed is between 20.1% and30%, or between 20.1% and 35%, preferably between 30% and 35%.

In another aspect, the present invention provides an oilseed plant orpart thereof such as a seed comprising

a) lipid in its seed, the lipid comprising fatty acids in an esterifiedform, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase        and an Δ5-elongase, or    -   ii) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase        and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or moreseed-specific promoters that are capable of directing expression of saidpolynucleotides in developing seed of the plant, wherein the fatty acidscomprise oleic acid, palmitic acid, ω6 fatty acids which compriselinoleic acid (LA) and γ-linolenic acid (GLA), ω3 fatty acids whichcomprise α-linolenic acid (ALA), stearidonic acid (SDA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), andoptionally eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid(ETA), and wherein the level of DHA in the total fatty acid content ofthe lipid of the seed is between 20.1% and 30%, or between 20.1% and35%, and wherein the level of palmitic acid in the total fatty acidcontent of the lipid is between about 2% and 16%, and wherein the levelof myristic acid (C14:0) in the total fatty acid content of the lipid,if present, is less than 1%.

In another aspect, the present invention provides an oilseed plant orpart thereof such as a seed comprising lipid in its seed, or a microbialcell, comprising

a) lipid comprising fatty acids in an esterified form, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an        Δ5-elongase,    -   ii) a Δ12-desaturase, a ω3-desaturase and/or Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase and an        Δ5-elongase,    -   iii) a ω3-desaturase and/or Δ15-desaturase, a Δ6-desaturase, a        Δ5-desaturase, a Δ6-elongase and an Δ5-elongase, or    -   iv) a ω3-desaturase and/or Δ15-desaturase, a Δ8-desaturase, a        Δ5-desaturase, a Δ9-elongase and an Δ5-elongase,

wherein each polynucleotide is operably linked to one or moreseed-specific promoters that are capable of directing expression of saidpolynucleotides in developing seed of the plant, or one or morepromoters that are capable of directing expression of saidpolynucleotides in the microbial cell, wherein the fatty acids compriseoleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid(LA) and optionally γ-linolenic acid (GLA), ω3 fatty acids whichcomprise α-linolenic acid (ALA), stearidonic acid (SDA), anddocosapentaenoic acid (DPA), and optionally docosahexaenoic acid (DHA),eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid (ETA), andwherein the level of DPA in the total fatty acid content of the lipid ofthe seed or microbial cell is between 7% and 35%. In a preferredembodiment of this aspect, DHA is present at a level of less than 0.5%of the total fatty acid content of the lipid of the seed and of theextracted lipid and more preferably is absent from the total fatty acidcontent of the lipids.

In another aspect, the present invention provides a cell, preferably acell in or from a plant such as an oilseed plant or part thereof such asa seed, or an oilseed plant or part thereof, or a microbial cell,comprising

a) fatty acids in an esterified form, the fatty acids comprisingdocosapentaenoic acid (DPA) and/or docosahexaenoic acid (DHA), whereinat least 35% of the DPA and/or DHA esterified in the form oftriacylglycerol (TAG) is esterified at the sn-2 position of the TAG, and

b) exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), an        ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase,        a Δ5-elongase and optionally a Δ4-desaturase,    -   ii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase,        a Δ5-elongase and optionally a Δ4-desaturase,    -   iii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase,        an Δ5-elongase and optionally a Δ4-desaturase,    -   iv) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an Δ5-elongase        and optionally a Δ4-desaturase,    -   v) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), an        ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase,        an Δ5-elongase and optionally a Δ4-desaturase,    -   vi) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase,        a Δ5-elongase and optionally a Δ4-desaturase,    -   vii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase,        an Δ5-elongase and optionally a Δ4-desaturase,    -   viii) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a        Δ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, an Δ5-elongase        and optionally a Δ4-desaturase,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of said polynucleotides in thecell. Preferably, the LPAAT can use a C22 polyunsaturated fatty acyl-CoAsubstrate such as DHA-CoA and/or DPA-CoA and the level of DPA and/or DHAin the total fatty acid content of the extracted lipid is between about1% and 35%, or between about 7% and 35% or between about 20.1% and 35%.In embodiments, at least about 40%, at least about 45%, at least about48%, between 35% and about 60%, or between 35% and about 50%, of the DPAand/or DHA esterified in the form of triacylglycerol (TAG) is esterifiedat the sn-2 position of the TAG.

In embodiments of each of the above five aspects, the Δ15-desaturase isa fungal Δ15-desaturase and the ω3-desaturase is a fungal ω3-desaturase.

In a preferred embodiment, the oilseed plant, microbial cell or cell ofthe invention has, where relevant, one or more of the features definedherein, for example as defined above in relation to extracted plantlipid, extracted microbial lipid or a process for the productionthereof.

Examples of oilseed plants include, but are not limited to, Brassicasp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamustinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghumbicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesisguineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinusangustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, orCrambe abyssinica. In an embodiment, the plant is a Brassica sp. plant,a C. sativa plant or a G. max (soybean) plant. In an embodiment, theoilseed plant is a canola, B. juncea, Glycine max, Camelina sativa orArabidopsis thaliana plant. In an alternate embodiment, the oilseedplant is other than A. thaliana and/or other than C. sativa. In anembodiment, the oilseed plant is a plant other than G. max (soybean). Inan embodiment, the oilseed plant is in the field, or was grown in thefield, or was grown in a glasshouse under standard conditions, forexample as described in Example 1.

In an embodiment, one or more of the desaturases used in a process ofthe invention or present in a cell, or plant or part thereof of theinvention, is capable of using an acyl-CoA substrate. In a preferredembodiment, one or more of the Δ6-desaturase, Δ5-desaturase,Δ4-desaturase and Δ8-desaturase, if present, is capable of using anacyl-CoA substrate, preferably each of the i) Δ6-desaturase,Δ5-desaturase and Δ4-desaturase or ii) Δ5-desaturase, Δ4-desaturase andΔ8-desaturase is capable of using an acyl-CoA substrate. In anembodiment, a Δ12-desaturase and/or an ω3-desaturase is capable of usingan acyl-CoA substrate. The acyl-CoA substrate is preferably an ALA-CoAfor Δ6-desaturase, ETA-CoA for Δ5-desaturase, DPA-CoA for Δ4-desaturase,and ETrA-CoA for Δ8-desaturase, oleoyl-CoA for the Δ12-desaturase, orone or more of LA-CoA, GLA-CoA, and ARA-CoA for ω3-desaturase.

In an embodiment, mature, harvested seed of the plant has a DHA and/orDPA content of at least about 28 mg per gram seed, preferably at leastabout 32 mg per gram seed, at least about 36 mg per gram seed, at leastabout 40 mg per gram seed, more preferably at least about 44 mg per gramseed or at least about 48 mg per gram seed, about 80 mg per gram seed,or between about 30 mg and about 80 mg per gram seed.

In a further aspect, the present invention provides a Brassica napus, B.juncea or Camelina sativa plant which is capable of producing seedcomprising DHA and/or DPA, wherein mature, harvested seed of the planthas a DHA and/or DPA content of at least about 28 mg per gram seed,preferably at least about 32 mg per gram seed, at least about 36 mg pergram seed, at least about 40 mg per gram seed, more preferably at leastabout 44 mg per gram seed or at least about 48 mg per gram seed, about80 mg per gram seed, or between about 30 mg and about 80 mg per gramseed.

In another aspect, the present invention provides a plant cell of aplant of the invention comprising the exogenous polynucleotides definedherein.

Also provided is a plant part, preferably a seed, or recombinant cellssuch as microbial cells which has one or more of the following features

i) is from a plant of the invention,

ii) comprises lipid as defined herein, or

iii) can be used in a process of the invention.

In yet another aspect, the present invention provides mature, harvestedBrassica napus, B. juncea or Camelina sativa seed comprising DHA and/orDPA and a moisture content of between about 4% and about 15% by weight,preferably between about 6% and about 8% by weight or between about 4%and about 8% by weight, more preferably between about 4% and about 6% byweight, wherein the DHA and/or DPA content of the seed is at least about28 mg per gram seed, preferably at least about 32 mg per gram seed, atleast about 36 mg per gram seed, at least about 40 mg per gram seed,more preferably at least about 44 mg per gram seed or at least about 48mg per gram seed, about 80 mg per gram seed, or between about 30 mg andabout 80 mg per gram seed.

In an embodiment, the cell of the invention, the oilseed plant of theinvention, the Brassica napus, B. juncea or Camelina sativa plant of theinvention, the plant part of the invention, or the seed of theinvention, can be used to produce extracted lipid comprising one or moreor all of the features defined herein.

In yet a further aspect, the present invention provides a method ofproducing a plant or cell which can be used to produce extracted lipidof the invention, the method comprising

a) assaying the level of DHA and/or DPA in lipid produced by one or moreplant parts such as seeds or recombinant cells such as microbial cellsfrom a plurality of plants or recombinant cells such as microbial cells,each plant or recombinant cell such as a microbial cell comprising oneor more exogenous polynucleotides encoding one of the following sets ofenzymes;

-   -   i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,    -   ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,    -   iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,    -   iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase        and an Δ5-elongase,    -   v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,    -   vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ9-elongase and a Δ5-elongase,    -   vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a        Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,    -   viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase        and an Δ5-elongase,    -   ix) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ6-elongase and a Δ5-elongase,    -   x) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ6-elongase and a Δ5-elongase,    -   xi) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a        Δ6-elongase and an Δ5-elongase,    -   xiii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a        Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an        Δ5-elongase, or    -   xiv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a        Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase and an        Δ5-elongase, wherein each polynucleotide is operably linked to        one or more promoters that are capable of directing expression        of said polynucleotides in a cell of a plant part or recombinant        cell, and

b) identifying a plant or recombinant cell, from the plurality of plantsor recombinant cells, which can be used to produce extracted plant lipidor cell lipid of the invention in one or more of its parts, and

c) optionally, producing progeny plants or recombinant cells from theidentified plant or recombinant cell, or seed therefrom.

In an embodiment, the plant or recombinant cell further comprises anexogenous polynucleotide encoding an LPAAT as defined herein.

Preferably, the progeny plant is at least a second or third generationremoved from the identified plant, and is preferably homozygous for theone or more polynucleotides. More preferably, the one or morepolynucleotides are present in the progeny plant at only a singleinsertion locus. That is, the invention provides such a method which canbe used as a screening method to identify a plant or seed therefrom froma plurality of transformed candidate plants or seeds, wherein theidentified plant or its progeny plant produces lipid of the invention,preferably in its seed. Such a plant or progeny plant or its seed isselected if it produces lipid of the invention, in particular having thespecified DHA level and/or DPA level, or is not selected if it does notproduce lipid of the invention.

In an embodiment, the exogenous polynucleotide(s) present in a cell suchas a microbial cell, or plant or part thereof as defined herein, becomestably integrated into the genome of the cell, plant or the plant partsuch as seed. Preferably, the exogenous polynucleotide(s) become stablyintegrated into the genome of the cell, plant or plant part such as seedat a single locus in the genome, and is preferably homozygous for theinsertion. More preferably, the plant, plant part or seed is furthercharacterised in that it is lacking exogenous polynucleotides other thanone or more T-DNA molecules. That is, no exogenous vector sequences areintegrated into the genome other than the T-DNA sequences.

In an embodiment, before step a) the method includes introducing the oneor more exogenous polynucleotides into one or more cells of the plant.

Also provided is a plant produced using a method of the invention, andseeds of such plants.

In an embodiment, the plant of the invention is both male and femalefertile, preferably has levels of both male and female fertility thatare at least 70% relative to, or preferably are about the same as, acorresponding wild-type plant. In an embodiment, the pollen produced bythe plant of the invention or the plant produced from the seed of theinvention is 90-100% viable as determined by staining with a viabilitystain. For example, the pollen viability may be assessed as described inExample 1.

In another aspect, the present invention provides a method of producingseed, the method comprising,

a) growing a plant of the invention, or a plant which produces a part ofthe invention, preferably in a field as part of a population of at least1000 or 2000 or 3000 such plants or in an area of at least 1 hectare or2 hectares or 3 hectares planted at a standard planting density,alternatively in a glasshouse under standard conditions,

b) harvesting seed from the plant or plants, and

c) optionally, extracting lipid from the seed, preferably to produce oilwith a total DHA yield and/or DPA yield of at least 60 kg or 70 kg or 80kg DHA and/or DPA/hectare.

In an embodiment, the plant, plant cell, plant part or seed, orrecombinant cell, of the invention has one or more of the followingfeatures

i) its oil is as defined herein, or

ii) the plant part or seed or recombinant cell is capable of being usedin a process of the invention.

For example, the seed can be used to produce a plant of the invention.The plant may be grown in the field or in a glasshouse under standardconditions, for example as described in Example 1.

In a further aspect, the present invention provides lipid, or oil,produced by, or obtained from, using the process of the invention, thecell of the invention, the oilseed plant of the invention, the Brassicasp., Brassica napus, B. juncea, G. max or Camelina sativa plant of theinvention, the plant part of the invention, the seed of the invention,or the plant, plant cell, plant part or seed of the invention.Preferably, the lipid or oil is purified to remove contaminants such asnucleic acid (DNA and/or RNA), protein and/or carbohydrate, or pigmentssuch as chlorophyll. The lipid or oil may also be purified to enrich theproportion of TAG, for example by removal of free fatty acids (FFA) orphospholipid.

In an embodiment, the lipid or oil is obtained by extraction of oil froman oilseed. Examples of oil from oilseeds include, but are not limitedto, canola oil (Brassica napus, Brassica rapa ssp.), mustard oil(Brassica juncea), other Brassica oil, sunflower oil (Helianthus annus),linseed oil (Linum usitatissimum), soybean oil (Glycine max), saffloweroil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotianatabacum), peanut oil (Arachis hypogaea), palm oil, cottonseed oil(Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Perseaamericana), olive oil (Olea europaea), cashew oil (Anacardiumoccidentale), macadamia oil (Macadamia intergrifolia), almond oil(Prunus amygdalus) or Arabidopsis seed oil (Arabidopsis thaliana).

In an embodiment, a cell (recombinant cell) of, or used in, theinvention is a microbial cell such as a cell suitable for fermentation,preferably an oleaginous microbial cell which is capable of accumulatingtriacylglycerols to a level of at least 25% on a weight basis. Preferredfermentation processes are anaerobic fermentation processes, as are wellknown in the art. Suitable fermenting cells, typically microorganismsare able to ferment, i.e., convert, sugars, such as glucose or maltose,directly or indirectly into the desired fatty acids. Examples offermenting microorganisms include fungal organisms, such as yeast. Asused herein, “yeast” includes Saccharomyces spp., Saccharomycescerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromycesspp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey,and preferably Yarrowia lipolytica.

In a further aspect, the present invention provides fatty acid producedby, or obtained from, using the process of the invention, the cell ofthe invention, the oilseed plant of the invention, the Brassica sp.,Brassica napus, B. juncea, G. max or Camelina sativa plant of theinvention, the plant part of the invention, the seed of the invention,or the plant, plant cell, plant part or seed of the invention.Preferably the fatty acid is DHA and/or DPA. The fatty acid may be in amixture of fatty acids having a fatty acid composition as describedherein, or may be enriched so that the fatty acid comprises at least 40%or at least 90% of the fatty acid content of the mixture. In anembodiment, the fatty acid is non-esterified. Alternatively, the fattyacid is esterified such as, for example, to a methyl, ethyl, propyl orbutyl group.

Also provided is seedmeal obtained from seed of the invention orobtained from a plant of the invention. Preferred seedmeal includes, butnot necessarily limited to, Brassica sp., Brassica napus, B. juncea,Camelina sativa or Glycine max seedmeal. In an embodiment, the seedmealcomprises an exogenous polynucleotide(s) and/or genentic constructs asdefined herein. In a preferred embodiment, the seedmeal retains some ofthe lipid or oil produced in the seed from which the seedmeal isobtained, but at a low level (for example, less than 2% by weight) afterextraction of most of the lipid or oil. The seedmeal may be used as ananimal feed or as an ingredient in food production.

In another aspect, the present invention provides a compositioncomprising one or more of the lipid or oil of the invention, the fattyacid of the invention, the cell according of the invention, the oilseedplant of the invention, the Brassica sp., Brassica napus, B. juncea,Glycine max or Camelina sativa plant of the invention, the plant part ofthe invention, the seed of the invention, or the seedmeal of theinvention. In embodiments, the composition comprises a carrier suitablefor pharmaceutical, food or agricultural use, a seed treatment compound,a fertiliser, another food or feed ingredient, or added protein orvitamins.

Also provided is feedstuffs, cosmetics or chemicals comprising one ormore of the lipid or oil of the invention, the fatty acid of theinvention, the cell according of the invention, the oilseed plant of theinvention, the Brassica sp., Brassica napus, B. juncea, Glycine max orCamelina sativa plant of the invention, the plant part of the invention,the seed of the invention, the seedmeal of the invention, or thecomposition of the invention.

In another aspect, the present invention provides a method of producinga feedstuff, the method comprising mixing one or more of the lipid oroil of the invention, the fatty acid of the invention, the cellaccording of the invention, the oilseed plant of the invention, theBrassica sp., Brassica napus, B. juncea, Glycine max or Camelina sativaplant of the invention, the plant part of the invention, the seed of theinvention, the seedmeal of the invention, or the composition of theinvention, with at least one other food ingredient. The method maycomprise steps of blending, cooking, baking, extruding, emulsifying orotherwise formulating the feedstuff, or packaging the feedstuff, or ofanalysing the amount of lipid or oil in the feedstuff.

In another aspect, the present invention provides a method of treatingor preventing a condition which would benefit from a PUFA, preferablyDHA and/or DPA, the method comprising administering to a subject one ormore of the lipid or oil of the invention, the fatty acid of theinvention, the cell according of the invention, the oilseed plant of theinvention, the Brassica sp., Brassica napus, B. juncea, Glycine max orCamelina sativa plant of the invention, the plant part of the invention,the seed of the invention, the seedmeal of the invention, thecomposition of the invention, or the feedstuff of the invention. In apreferred embodiment, the PUFA is administered in the form of apharmaceutical composition comprising an ethyl ester of the PUFA. Thesubject may be a human or an animal other than a human.

Examples of conditions which would benefit from a PUFA include, but arenot limited to, elevated serum triglyceride levels, elevated serumcholesterol levels such as elevated LDL cholesterol levels, cardiacarrhythmia's, angioplasty, inflammation, asthma, psoriasis,osteoporosis, kidney stones, AIDS, multiple sclerosis, rheumatoidarthritis, Crohn's disease, schizophrenia, cancer, foetal alcoholsyndrome, attention deficient hyperactivity disorder, cystic fibrosis,phenylketonuria, unipolar depression, aggressive hostility,adrenoleukodystophy, coronary heart disease, hypertension, diabetes,obesity, Alzheimer's disease, chronic obstructive pulmonary disease,ulcerative colitis, restenosis after angioplasty, eczema, high bloodpressure, platelet aggregation, gastrointestinal bleeding,endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronicfatigue after viral infections or an ocular disease.

Also provided is the use of one or more of the lipid or oil of theinvention, the fatty acid of the invention, the cell according of theinvention, the oilseed plant of the invention, the Brassica sp.,Brassica napus, B. juncea, Glycine max or Camelina sativa plant of theinvention, the plant part of the invention, the seed of the invention,the seedmeal of the invention, the composition of the invention, or thefeedstuff of the invention for the manufacture of a medicament fortreating or preventing a condition which would benefit from a PUFApreferably DHA and/or DPA.

The production of the medicament may comprise mixing the oil of theinvention with a pharmaceutically acceptable carrier, for treatment of acondition as described herein. The method may comprise firstly purifyingthe oil and/or transesterification, and/or fractionation of the oil toincrease the level of DHA and/or DPA. In a particular embodiment, themethod comprises treating the lipid or oil such as canola oil to convertthe fatty acids in the oil to alkyl esters such as methyl or ethylesters. Further treatment such as fractionation or distillation may beapplied to enrich the lipid or oil for the DHA and/or DPA. In apreferred embodiment, the medicament comprises ethyl esters of DHAand/or DPA. In an even more preferred embodiment, the level of ethylesters of DHA and/or DPA in the medicament is between 30% and 50%, or atleast 80% or at least about 85% or at least 90% or at least about 95%.The medicament may further comprise ethyl esters of EPA, such as between30% and 50%, or at least 90%, of the total fatty acid content in themedicament. Such medicaments are suitable for administration to human oranimal subjects for treatment of medical conditions as described herein.

In another aspect, the present invention provides a method of tradingseed, comprising obtaining seed of the invention, and trading theobtained seed for pecuniary gain.

In an embodiment, obtaining the seed comprises cultivating plants of theinvention and/or harvesting the seed from the plants.

In another embodiment, obtaining the seed further comprises placing theseed in a container and/or storing the seed.

In a further embodiment, obtaining the seed further comprisestransporting the seed to a different location.

In yet another embodiment, the method further comprises transporting theseed to a different location after the seed is traded.

In a further embodiment, the trading is conducted using electronic meanssuch as a computer.

In yet a further aspect, the present invention provides a process ofproducing bins of seed comprising:

a) swathing, windrowing and/or reaping above-ground parts of plantscomprising seed of the invention,

b) threshing and/or winnowing the parts of the plants to separate theseed from the remainder of the plant parts, and

c) sifting and/or sorting the seed separated in step b), and loading thesifted and/or sorted seed into bins, thereby producing bins of seed.

In an embodiment, where relevant, the lipid or oil, preferably seedoil,of, or useful for, the invention has fatty levels about those providedin a Table in the Examples section, such as seed CT136-27-18-2 orCT136-27-18-19 of Table 10, or the seedoil of Tables 12, 20, 22, 23 or24.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Aerobic DHA biosynthesis pathways.

FIG. 2. Map of the T-DNA insertion region between the left and rightborders of pJP3416-GA7. RB denotes right border; LB, left border; TER,transcription terminator/polyadenylation region; PRO, promoter; Codingregions are indicated above the arrows, promoters and terminators belowthe arrows. Micpu-Δ6D, Micromonas pusilla Δ6-desaturase; Pyrco-ME,Pyramimonas cordata Δ6-elongase; Paysa-Δ5D, Pavlova salinaΔ5-desaturase; Picpa-ω3D, Pichia pastoris ω3-desaturase; Paysa-Δ4D, P.salina Δ4-desaturase; Lackl-Δ12D, Lachancea kluyveri Δ12-desaturase;Pyrco-Δ5E, Pyramimonas cordata Δ5-elongase. NOS denotes theAgrobacterium tumefaciens nopaline synthase transcriptionterminator/polyadenylation region; FP1, Brassica napus truncated napinpromoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine maxlectin transcription terminator/polyadenylation region; Cnl1 and Cnl2denotes the Linum usitatissimum conlinin1 or conlinin2 promoter orterminator. MAR denotes the Rb7 matrix attachment region from Nicotianatabacum.

FIG. 3. Map of the T-DNA insertion region between the left and rightborders of pJP3404. Labels are as in FIG. 2.

FIG. 4. Oil content (w/w) vs. DHA content, as a percentage of totalfatty acid content of lipid from transgenic Arabidopsis thaliana seeds.

FIG. 5. Positional distribution analysis by NMR on A) Tuna oil and, B)transgenic DHA Arabidopsis seed oil. The peaks labelled ‘DHA-alpha’represent the amount of DHA present at the sn-1 and sn-3 positions ofTAG (with no positional preference this would equal 66% of total DHA)whilst the peaks labelled ‘DHA-beta’ represent the amount of DHA presentat the sn-2 position of TAG (with no preference this would equal 33% ofDHA).

FIG. 6. LC-MS analysis of major DHA-containing triacylglycerol speciesin transgenic A. thaliana developing (grey) and mature (black) seeds.The number following the DHA denotes the total number of carbon atomsand total number of double bonds in the other two fatty acids. ThereforeDHA/34:1 can also be designated TAG 56:7, etc.

FIG. 7. (A) Basic phytosterol structure with ring and side chainnumbering. (B) Chemical structures of some of the phytosterols.

FIG. 8. Phylogenetic tree of known LPAATs.

FIG. 9. The various acyl exchange enzymes which transfer fatty acidsbetween PC, CoA pools, and TAG pools. Adapted from Singh et al. (2005).

FIG. 10. DHA levels in the total fatty acid content of seedoil obtainedfrom individual T2 seeds from B. napus seeds transformed with the T-DNAfrom the GA7-modB construct. Each dot shows the DHA level in anindividual seed, with each column of dots representing T2 seeds from anindividual T1 plant.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—pJP3416-GA7 nucleotide sequence.

SEQ ID NO:2—pGA7-mod_B nucleotide sequence.

SEQ ID NO:3—Codon-optimized open reading frame for expression ofLachancea kluyveri Δ12 desaturase in plants.

SEQ ID NO:4—Lachancea kluyveri Δ12-desaturase.

SEQ ID NO:5—Codon-optimized open reading frame for expression of Pichiapastoris ω3 desaturase in plants.

SEQ ID NO:6—Pichia pastoris ω3 desaturase.

SEQ ID NO:7—Open reading frame encoding Micromonas pusillaΔ6-desaturase.

SEQ ID NO:8—Codon-optimized open reading frame for expression ofMicromonas pusilla Δ6-desaturase in plants.

SEQ ID NO:9—Micromonas pusilla Δ6-desaturase.

SEQ ID NO:10—Open reading frame encoding Ostreococcus lucimarinusdesaturase.

SEQ ID NO:11—Codon-optimized open reading frame for expression ofOstreococcus lucimarinus Δ6-desaturase in plants.

SEQ ID NO:12—Ostreococcus lucimarinus Δ6-desaturase.

SEQ ID NO:13—Ostreococcus tauri Δ6-desaturase.

SEQ ID NO:14—Open reading frame encoding Pyramimonas cordataΔ6-elongase.

SEQ ID NO:15—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ6-elongase in plants (truncated at 3′ end andencoding functional elongase).

SEQ ID NO:16—Pyramimonas cordata Δ6-elongase.

SEQ ID NO:17—Truncated Pyramimonas cordata Δ6-elongase.

SEQ ID NO:18—Open reading frame encoding Pavlova salina Δ5-desaturase.

SEQ ID NO:19—Codon-optimized open reading frame for expression ofPavlova salina Δ5-desaturase in plants.

SEQ ID NO:20—Pavlova salina Δ6-desaturase.

SEQ ID NO:21—Open reading frame encoding Pyramimonas cordataΔ5-desaturase.

SEQ ID NO:22—Pyramimonas cordata Δ5-desaturase.

SEQ ID NO:23—Open reading frame encoding Pyramimonas cordataΔ5-elongase.

SEQ ID NO:24—Codon-optimized open reading frame for expression ofPyramimonas cordata Δ5-elongase in plants.

SEQ ID NO:25—Pyramimonas cordata Δ5-elongase.

SEQ ID NO:26—Open reading frame encoding Pavlova salina Δ4-desaturase.

SEQ ID NO:27—Codon-optimized open reading frame for expression ofPavlova salina Δ4-desaturase in plants.

SEQ ID NO:28—Pavlova salina Δ4-desaturase.

SEQ ID NO:29—Isochrysis galbana Δ9-elongase.

SEQ ID NO:30—Codon-optimized open reading frame for expression ofEmiliania huxleyi Δ9-elongase in plants.

SEQ ID NO:31—Emiliania huxleyi CCMP1516 Δ9-elongase.

SEQ ID NO:32—Open reading frame encoding Pavlova pinguis Δ9-elongase.

SEQ ID NO:33—Pavlova pinguis Δ9-elongase.

SEQ ID NO:34—Open reading frame encoding Pavlova salina Δ9-elongase.

SEQ ID NO:35—Pavlova salina Δ9-elongase.

SEQ ID NO:36—Open reading frame encoding Pavlova salina Δ8-desaturase.

SEQ ID NO:37—Pavlova salina Δ8-desaturase.

SEQ ID NO:38—V2 viral suppressor.

SEQ ID NO:39—Open reading frame encoding V2 viral suppressor.

SEQ ID NO: 40—Arabidopsis thaliana LPAAT2.

SEQ ID NO: 41—Limnanthes alba LPAAT.

SEQ ID NO: 42—Saccharomyces cerevisiae LPAAT.

SEQ ID NO: 43—Micromonas pusilla LPAAT.

SEQ ID NO: 44—Mortierella alpina LPAAT.

SEQ ID NO: 45—Braccisa napus LPAAT.

SEQ ID NO: 46—Brassica napus LPAAT.

SEQ ID NO: 47—Phytophthora infestans ω3 desaturase.

SEQ ID NO: 48—Thalassiosira pseudonana ω3 desaturase.

SEQ ID NO: 49—Pythium irregulare ω3 desaturase.

SEQ ID NO's: 50 to 58—Oligonucleotide primers/probes.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, fatty acid synthesis, transgenic plants, recombinantcells, protein chemistry, and biochemistry).

Unless otherwise indicated, the protein, cell culture, and immunologicaltechniques utilized in the present invention are standard procedures,well known to those skilled in the art. Such techniques are describedand explained throughout the literature in sources such as, J. Perbal, APractical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors), Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors), Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about” unless stated to the contrary, refersto +/−10%, more preferably +/−5%, more preferably +/−1% of thedesignated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Selected Definitions

As used herein, the terms “extracted plant lipid” and “isolated plantlipid” refer to a lipid composition which has been extracted from, forexample by crushing, a plant or part thereof such as seed. The extractedlipid can be a relatively crude composition obtained by, for example,crushing a plant seed, or a more purified composition where most, if notall, of one or more or each of the water, nucleic acids, proteins andcarbohydrates derived from the plant material have been removed.Examples of purification methods are described below. In an embodiment,the extracted or isolated plant lipid comprises at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 95% (w/w) lipid by weight of the composition. The lipid may besolid or liquid at room temperature, when liquid it is considered to bean oil. In an embodiment, extracted lipid of the invention has not beenblended with another lipid such as DHA and/or DPA produced by anothersource (for example, DHA from fish oil). In an embodiment, followingextraction the ratio of one or more or all of, oleic acid to DHA and/orDPA, palmitic acid to DHA and/or DPA, linoleic acid to DHA and/or DPA,and total ω6 fatty acids: total ω3 fatty acids, has not beensignificantly altered (for example, no greater than a 10% or 5%alteration) when compared to the ratio in the intact seed or cell. In ananother embodiment, the extracted plant lipid has not been exposed to aprocedure, such as hydrogenation or fractionation, which may alter theratio of one or more or all of, oleic acid to DHA and/or DPA, palmiticacid to DHA and/or DPA, linoleic acid to DHA and/or DPA, and total ω6fatty acids: total ω3 fatty acids, when compared to the ratio in theintact seed or cell. When the extracted plant lipid of the invention iscomprised in an oil, the oil may further comprise non-fatty acidmolecules such as sterols.

As used herein, the terms “extracted plant oil” and “isolated plant oil”refer to a substance or composition comprising extracted plant lipid orisolated plant lipid and which is a liquid at room temperature. The oilis obtained from a plant or part thereof such as seed. The extracted orisolated oil can be a relatively crude composition obtained by, forexample, crushing a plant seed, or a more purified composition wheremost, if not all, of one or more or each of the water, nucleic acids,proteins and carbohydrates derived from the plant material have beenremoved. The composition may comprise other components which may belipid or non-lipid. In an embodiment, the oil composition comprises atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 95% (w/w) extracted plant lipid. In anembodiment, extracted oil of the invention has not been blended withanother oil such as DHA and/or DPA produced by another source (forexample, DHA from fish oil). In an embodiment, following extraction, theratio of one or more or all of, oleic acid to DHA and/or DPA, palmiticacid to DHA and/or DPA, linoleic acid to DHA and/or DPA, and total ω6fatty acids: total ω3 fatty acids, has not been significantly altered(for example, no greater than a 10% or 5% alteration) when compared tothe ratio in the intact seed or cell. In an another embodiment, theextracted plant oil has not been exposed to a procedure, such ashydrogenation or fractionation, which may alter the ratio of one or moreor all of, oleic acid to DHA and/or DPA, palmitic acid to DHA and/orDPA, linoleic acid to DHA and/or DPA, and total ω6 fatty acids: total ω3fatty acids, when compared to the ratio in the intact seed or cell.Extracted plant oil of the invention may comprise non-fatty acidmolecules such as sterols.

As used herein, terms such as “extracted microbial lipid” or “extractedmicrobial oil” have analogous meanings as the corresponding terms“extracted plant lipid” and “extracted plant oil” respectively, with themain difference being the source of the lipid or oil.

As used herein, an “oil” is a composition comprising predominantly lipidand which is a liquid at room temperature. For instance, oil of theinvention preferably comprises at least 75%, at least 80%, at least 85%or at least 90% lipid by weight. Typically, a purified oil comprises atleast 90% triacylglycerols (TAG) by weight of the lipid in the oil.Minor components of an oil such as diacylglycerols (DAG), free fattyacids (FFA), phospholipid and sterols may be present as describedherein.

As used herein, the term “fatty acid” refers to a carboxylic acid (ororganic acid), often with a long aliphatic tail, either saturated orunsaturated. Typically fatty acids have a carbon-carbon bonded chain ofat least 8 carbon atoms in length, more preferably at least 12 carbonsin length. Preferred fatty acids of the invention have carbon chains of18-22 carbon atoms (C18, C20, C22 fatty acids), more preferably 20-22carbon atoms (C20, C22) and most preferably 22 carbon atoms (C22). Mostnaturally occurring fatty acids have an even number of carbon atomsbecause their biosynthesis involves acetate which has two carbon atoms.The fatty acids may be in a free state (non-esterified) or in anesterified form such as part of a triglyceride, diacylglyceride,monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. Thefatty acid may be esterified as a phospholipid such as aphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerolforms. In an embodiment, the fatty acid is esterified to a methyl orethyl group, such as, for example, a methyl or ethyl ester of a C20 orC22 PUFA. Preferred fatty acids are the methyl or ethyl esters of EPA,DPA or DHA, or the mixtures EPA and DHA, or EPA, DPA and DHA, or EPA andDPA.

“Saturated fatty acids” do not contain any double bonds or otherfunctional groups along the chain. The term “saturated” refers tohydrogen, in that all carbons (apart from the carboxylic acid [—COOH]group) contain as many hydrogens as possible. In other words, the omega(ω) end contains 3 hydrogens (—CH3-) and each carbon within the chaincontains 2 hydrogens (—CH2-).

“Unsaturated fatty acids” are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration, preferably in the cis configuration. In an embodiment,the lipid or oil or the invention has a fatty acid composition whichcomprises less than 1% fatty acids having a carbon-carbon double bond inthe trans configuration (trans fatty acids).

As used herein, the term “monounsaturated fatty acid” refers to a fattyacid which comprises at least 12 carbon atoms in its carbon chain andonly one alkene group (carbon-carbon double bond) in the chain. As usedherein, the terms “polyunsaturated fatty acid” or “PUFA” refer to afatty acid which comprises at least 12 carbon atoms in its carbon chainand at least two alkene groups (carbon-carbon double bonds). As usedherein, the terms “long-chain polyunsaturated fatty acid” and “LC-PUFA”refer to a fatty acid which comprises at least 20 carbon atoms in itscarbon chain and at least two carbon-carbon double bonds, and henceinclude VLC-PUFAs. As used herein, the terms “very long-chainpolyunsaturated fatty acid” and “VLC-PUFA” refer to a fatty acid whichcomprises at least 22 carbon atoms in its carbon chain and at leastthree carbon-carbon double bonds. Ordinarily, the number of carbon atomsin the carbon chain of the fatty acids refers to an unbranched carbonchain. If the carbon chain is branched, the number of carbon atomsexcludes those in sidegroups. In one embodiment, the long-chainpolyunsaturated fatty acid is an ω3 fatty acid, that is, having adesaturation (carbon-carbon double bond) in the third carbon-carbon bondfrom the methyl end of the fatty acid. In another embodiment, thelong-chain polyunsaturated fatty acid is an ω6 fatty acid, that is,having a desaturation (carbon-carbon double bond) in the sixthcarbon-carbon bond from the methyl end of the fatty acid. In a furtherembodiment, the long-chain polyunsaturated fatty acid is selected fromthe group consisting of; arachidonic acid (ARA, 20:4Δ5,8,11,14; ω6),eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3), eicosapentaenoic acid(EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA,22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA,22:6Δ4,7,10,13,16,19, ω3). The LC-PUFA may also be dihomo-γ-linoleicacid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11,14,17, ω3). It wouldreadily be apparent that the LC-PUFA that is produced according to theinvention may be a mixture of any or all of the above and may includeother LC-PUFA or derivatives of any of these LC-PUFA. In a preferredembodiment, the ω3 fatty acids are at least DHA and/or DPA, preferably,DPA and DHA, or EPA, DPA and DHA, or EPA and DPA. As extracted from theplant, DHA is present in the lipid or oil at a level of 20.1-30% orbetween 20.1% and 35%, preferably between 30% to 35% of the total fattyacid composition. For example, DHA is present at a level of between30.1% and 35% of the total fatty acid composition. In an embodiment, thelevel of DHA is greater than the level of DPA, more preferably greaterthan the level of each of EPA and DPA, most preferably greater than thecombined level of EPA and DPA. In an alternative embodiment, DPA ispresent at a level of between about 7% and 30% or 35% and DHA is eitherabsent or, if present, is present at a level of less than 2.0%,preferably less than 1.0%, more preferably less than 0.5% of the totalfatty acid composition and most preferably absent or undetectable. Thismay be accomplished by the absence of a Δ4-desaturase activity in thecell. In an embodiment, the level of DPA is greater than the level ofEPA, more preferably greater than the level of each of EPA and DHA, mostpreferably greater than the combined level of EPA and DHA. In thisembodiment, DHA may be absent or, if present, is present at a level ofless than 0.5% of the total fatty acid composition.

Furthermore, as used herein the terms “long-chain polyunsaturated fattyacid” (LC-PUFA) and “very long-chain polyunsaturated fatty acid”(VLC-PUFA) refer to the fatty acid being in a free state(non-esterified) or in an esterified form such as part of a triglyceride(triacylglycerol), diacylglyceride, monoacylglyceride, acyl-CoA bound orother bound form. In the triglyceride, the LC-PUFA or VLC-PUFA such asDHA or DPA may be esterified at the sn-1/3 or sn-2 positions, or thetriglyceride may comprise two or three acyl groups selected from LC-PUFAand VLC-PUFA acyl groups. For example, the triglyceride may comprise DHAor DPA at both of the sn-1 and sn-3 positions. The fatty acid may beesterified as a phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol or diphosphatidylglycerol forms. Thus, the LC-PUFAmay be present as a mixture of forms in the lipid of a cell or apurified oil or lipid extracted from cells, tissues or organisms. Inpreferred embodiments, the invention provides oil comprising at least75% or at least 85% triacylglycerols, with the remainder present asother forms of lipid such as those mentioned, with at least saidtriacylglycerols comprising the LC-PUFA. The oil may subsequently befurther purified or treated, for example by hydrolysis with a strongbase to release the free fatty acids, or by transesterification,distillation or the like.

As used herein, “total ω6 fatty acids” or “total ω6 fatty acid content”or the like refers to the sum of all the ω6 fatty acids, esterified andnon-esterified, in the extracted lipid, oil, recombinant cell, plantpart or seed, as the context determines, expressed as a percentage ofthe total fatty acid content. These ω6 fatty acids include (if present)LA, GLA, DGLA, ARA, EDA and ω6-DPA, and exclude any ω3 fatty acids andmonounsaturated fatty acids. The ω6 fatty acids present in the plants,seeds, lipid or oils of the invention are all included in the class ofpolyunsaturated fatty acids (PUFA).

As used herein, “new ω6 fatty acids” or “new ω6 fatty acid content” orthe like refers to the sum of all the ω6 fatty acids excluding LA,esterified and non-esterified, in the extracted lipid, oil, recombinantcell, plant part or seed, as the context determines, expressed as apercentage of the total fatty acid content. These new ω6 fatty acids arethe fatty acids that are produced in the cells, plants, plant parts andseeds of the invention by the expression of the genetic constructs(exogenous polynucleotides) introduced into the cells, and include (ifpresent) GLA, DGLA, ARA, EDA and ω6-DPA, but exclude LA and any ω3 fattyacids and monounsaturated fatty acids. Exemplary total ω6 fatty acidcontents and new ω6 fatty acid contents are determined by conversion offatty acids in a sample to FAME and analysis by GC, as described inExample 1.

As used herein, “total ω3 fatty acids” or “total ω3 fatty acid content”or the like refers to the sum of all the ω3 fatty acids, esterified andnon-esterified, in the extracted lipid, oil, recombinant cell, plantpart or seed, as the context determines, expressed as a percentage ofthe total fatty acid content. These ω3 fatty acids include (if present)ALA, SDA, ETrA, ETA, EPA, DPA and DHA, and exclude any ω6 fatty acidsand monounsaturated fatty acids. The ω3 fatty acids present in theplants, seeds, lipid or oils of the invention are all included in theclass of polyunsaturated fatty acids (PUFA).

As used herein, “new ω3 fatty acids” or “new ω3 fatty acid content” orthe like refers to the sum of all the ω3 fatty acids excluding ALA,esterified and non-esterified, in the extracted lipid, oil, recombinantcell, plant part or seed, as the context determines, expressed as apercentage of the total fatty acid content. These new ω3 fatty acids arethe ω3 fatty acids that are produced in the cells, plants, plant partsand seeds of the invention by the expression of the genetic constructs(exogenous polynucleotides) introduced into the cells, and include (ifpresent) SDA, ETrA, ETA, EPA, DPA and DHA, but exclude ALA and any ω6fatty acids and monounsaturated fatty acids. Exemplary total ω3 fattyacid contents and new ω3 fatty acid contents are determined byconversion of fatty acids in a sample to FAME and analysis by GC, asdescribed in Example 1.

As the skilled person would appreciate, the term “obtaining a plantpart” as a step in the process of the invention can include obtainingone or more plant parts for use in the process. Obtaining the plant partincludes harvesting the plant part from a plant such as with amechanical harvester, or purchasing the plant part, or receiving theplant part from a supplier. In another example, obtaining a plant partmay be acquiring the plant from someone else who has harvested the plantpart.

The desaturase, elongase and acyl transferase proteins and genesencoding them that may be used in the invention are any of those knownin the art or homologues or derivatives thereof. Examples of such genesand encoded protein sizes are listed in Table 1. The desaturase enzymesthat have been shown to participate in LC-PUFA biosynthesis all belongto the group of so-called “front-end” desaturases. Preferred proteins,or combinations of proteins, are those encoded by the genetic constructsprovided herein as SEQ ID NOs: 1 and 2.

TABLE 1 Cloned genes involved in LC-PUFA biosynthesis Protein sizeEnzyme Type of organism Species Accession Nos. (aa's) ReferencesΔ4-desaturase Protist Euglena gracilis AY278558 541 Meyer et al., 2003Algae Pavlova lutherii AY332747 445 Tonon et al., 2003 Isochrysisgalbana AAV33631 433 Pereira et al., 2004b Pavlova salina AAY15136 447Thou et al., 2007 Thraustochytrid Thraustochytrium aureum AAN75707 515N/A AAN75708 AAN75709 AAN75710 Thraustochytrium sp. AAM09688 519 Qiu etal. 2001 ATCC21685 Δ5-desaturase Mammals Homo sapiens AF199596 444 Choet al., 1999b Leonard et al., 2000b Nematode Caenorhabditis elegansAF11440, 447 Michaelson et al., 1998b; NM_069350 Watts and Browse, 1999bFungi Mortierella alpina AF067654 446 Michaelson et al., 1998a; Knutzonet al., 1998 Pythium irregulare AF419297 456 Hong et al., 2002aDictyostelium discoideum AB022097 467 Saito et al., 2000 Saprolegniadiclina 470 WO02081668 Diatom Phaeodactylum tricornutum AY082392 469Domergue et al., 2002 Algae Thraustochytrium sp AF489588 439 Qiu et al.,2001 Thraustochytrium aureum 439 WO02081668 Isochrysis galbana 442WO02081668 Moss Marchantia polymorpha AY583465 484 Kajikawa et al., 2004Δ6-desaturase Mammals Homo sapiens NM_013402 444 Cho et al., 1999a;Leonard et al., 2000 Mus musculus NM_019699 444 Cho et al., 1999aNematode Caenorhabditis elegans Z70271 443 Napier et al., 1998 PlantsBorago officinales U79010 448 Sayanova et al., 1997 Echium AY055117Garcia-Maroto et al., 2002 AY055118 Primula vialii AY234127 453 Sayanovaet al., 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 MossesCeratodon purpureus AJ250735 520 Sperling et al., 2000 Marchantiapolymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patensCAA11033 525 Girke et al., 1998 Fungi Mortierella alpina AF110510 457Huang et al., 1999; A8020032 Sakuradani et al., 1999 Pythium irregulareAF419296 459 Hong et al., 2002a Mucor circinelloides AB052086 467 NCBI*Rhizopus sp. AY320288 458 Zhang et al., 2004 Saprolegnia diclina 453WO02081668 Diatom Phaeodactylum tricornutum AY082393 477 Domergue etal., 2002 Bacteria Synechocystis L11421 359 Reddy et al., 1993 AlgaeThraustochytrium aureum 456 WO02081668 Bifunctional Fish Danio rerioAF309556 444 Hastings et al., 2001 Δ5/Δ6-desaturase C20 Δ8- AlgaeEuglena gracilis AF139720 419 Wallis and Browse, 1999 desaturase PlantsBorago officinales AAG43277 446 Sperling et al., 2001 Δ6-elongaseNematode Caenorhabditis elegans NM_069288 288 Beaudoin et al., 2000Mosses Physcomitrella patens AF428243 290 Zank et al., 2002 Marchantiapolymorpha AY583464 290 Kajikawa et al., 2004 Fungi Mortierella alpinaAF206662 318 Parker-Barnes et al., 2000 Algae Pavlova lutheri** 501 WO03078639 Thraustochytrium AX951565 271 WO 03093482 Thraustochytrium sp**AX214454 271 WO 0159128 PUFA- Mammals Homo sapiens AF231981 299 Leonardet al., 2000b; elongase Leonard et al., 2002 Rattus norvegicus AB071985299 Inagaki et al., 2002 Rattus norvegicus** AB071986 267 Inagaki etal., 2002 Mus musculus AF170907 279 Tvrdik et al., 2000 Mus musculusAF170908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291 (282)Agaba et al., 2004 Danio rerio** NM_199532 266 Lo et al., 2003 WormCaenorhabditis elegans Z68749 309 Abbott et al., 1998 Beaudoin et al.,2000 Algae Thraustochytrium aureum** AX464802 272 WO 0208401-A2 Pavlovalutheri** 320 WO 03078639 Δ9-elongase Algae Isochrysis galbana AF390174263 Qi et al., 2002 Euglena gracilis 258 WO 08/128241 Δ5-elongase AlgaeOstreococcus tauri AAV67798 300 Meyer et al., 2004 Pyramimonas cordata268 WO 2010/057246 Pavlova sp. CCMP459 AAV33630 277 Pereira et al.,2004b Pavlova salina AAY15135 302 Robert et al., 2009 DiatomThalassiosira pseudonana AAV67800 358 Meyer et al., 2004 FishOncorhynchus mykiss CAM55862 295 WO 06/008099 Moss Marchantia polymorphaBAE71129 348 Kajikawa et al., 2006  *http://www.ncbi.nlm.nili.gov/**Function not proven/not demonstrated

As used herein, the term “front-end desaturase” refers to a member of aclass of enzymes that introduce a double bond between the carboxyl groupand a pre-existing unsaturated part of the acyl chain of lipids, whichare characterized structurally by the presence of an N-terminalcytochrome b5 domain, along with a typical fatty acid desaturase domainthat includes three highly conserved histidine boxes (Napier et al.,1997).

Activity of any of the elongases or desaturases for use in the inventionmay be tested by expressing a gene encoding the enzyme in a cell suchas, for example, a plant cell or preferably in somatic embryos ortransgenic plants, and determining whether the cell, embryo or plant hasan increased capacity to produce LC-PUFA compared to a comparable cell,embryo or plant in which the enzyme is not expressed.

In one embodiment one or more of the desaturases and/or elongases foruse in the invention can purified from a microalga, i.e. is identical inamino acid sequence to a polypeptide which can be purified from amicroalga.

Whilst certain enzymes are specifically described herein as“bifunctional”, the absence of such a term does not necessarily implythat a particular enzyme does not possess an activity other than thatspecifically defined.

Desaturases

As used herein, the term “desaturase” refers to an enzyme which iscapable of introducing a carbon-carbon double bond into the acyl groupof a fatty acid substrate which is typically in an esterified form suchas, for example, acyl-CoA esters. The acyl group may be esterified to aphospholipid such as phosphatidylcholine (PC), or to acyl carrierprotein (ACP), or in a preferred embodiment to CoA. Desaturasesgenerally may be categorized into three groups accordingly. In oneembodiment, the desaturase is a front-end desaturase.

As used herein, a “Δ4-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the4^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. The “Δ4-desaturase” is at least capable of converting DPA toDHA. Preferably, the “Δ4-desaturase” is capable of converting DPA-CoA toDHA-CoA, i.e. it is an acyl CoA desaturase. In an embodiment, the“Δ4-desaturase” is capable of converting DPA esterified at the sn-2position of PC to DHA-PC. Preferably the Δ4-desaturase has greateractivity on DPA-CoA than on DPA-PC. The desaturation step to produce DHAfrom DPA is catalysed by a Δ4-desaturase in organisms other thanmammals, and a gene encoding this enzyme has been isolated from thefreshwater protist species Euglena gracilis and the marine speciesThraustochytrium sp. (Qiu et al., 2001; Meyer et al., 2003). In oneembodiment, the Δ4-desaturase comprises amino acids having a sequence asprovided in SEQ ID NO:28, or a Thraustochytrium sp. Δ4-desaturase, abiologically active fragment thereof, or an amino acid sequence which isat least 80% identical to SEQ ID NO:28. In an embodiment, a plant, plantpart (such as seed) or cell of, or used in, the invention which produceshigh levels of DPA, such as 5% to 35% of the total extractable fattyacid content is DPA, does not comprise a gene encoding a functionalΔ4-desaturase.

As used herein, a “Δ5-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the5^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. In an embodiment, the fatty acid substrate is ETA and theenzyme produces EPA. Preferably, the “Δ5-desaturase” is capable ofconverting ETA-CoA to EPA-CoA, i.e. it is an acyl-CoA desaturase. In anembodiment, the “Δ5-desaturase” is capable of converting ETA esterifiedat the sn-2 position of PC. Preferably the Δ5-desaturase has greateractivity on ETA-CoA than on ETA-PC. Examples of Δ5-desaturases arelisted in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and inTable 1 herein. In one embodiment, the Δ5-desaturase comprises aminoacids having a sequence as provided in SEQ ID NO:20, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 80%identical to SEQ ID NO:20. In another embodiment, the Δ5-desaturasecomprises amino acids having a sequence as provided in SEQ ID NO:22, abiologically active fragment thereof, or an amino acid sequence which isat least 53% identical to SEQ ID NO:22. In another embodiment, theΔ5-desaturase is from Thraustochytrium sp or Emiliania huxleyi.

As used herein, a “Δ6-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the6^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. In an embodiment, the fatty acid substrate is ALA and theenzyme produces SDA. Preferably, the “Δ6-desaturase” is capable ofconverting ALA-CoA to SDA-CoA, i.e. it is an acyl-CoA desaturase. In anembodiment, the “Δ6-desaturase” is capable of converting ALA esterifiedat the sn-2 position of PC. Preferably the Δ6-desaturase has greateractivity on ALA-CoA than on ALA-PC. The Δ6-desaturase may also haveactivity as a Δ5-desaturase, being termed a Δ5/Δ6 bifunctionaldesaturase, so long as it has greater Δ6-desaturase activity on ALA thanΔ5-desaturase activity on ETA. Examples of Δ6-desaturases are listed inRuiz-Lopez et al. (2012) and Petrie et al. (2010a) and in Table 1herein. Preferred Δ6-desaturases are from Micromonas pusilla, Pythiumirregulare or Ostreococcus taurii.

In an embodiment, the Δ6-desaturase is further characterised by havingat least two, preferably all three and preferably in a plant cell, ofthe following: i) greater Δ6-desaturase activity on α-linolenic acid(ALA, 18:3Δ9,12,15, ω3) than linoleic acid (LA, 18:2Δ9,12, ω6) as fattyacid substrate; ii) greater Δ6-desaturase activity on ALA-CoA as fattyacid substrate than on ALA joined to the sn-2 position of PC as fattyacid substrate; and iii) Δ8-desaturase activity on ETrA. Examples ofsuch Δ6-desaturases are provided in Table 2.

TABLE 2 Desaturases demonstrated to have activity on an acyl-CoAsubstrate Protein Type of Accession size Enzyme organism Species Nos.(aa's) References Δ6-desaturase Algae Mantoniella CAQ30479 449 Hoffmannet al., 2008 squamata Ostreococcus AAW70159 456 Domergue et al., 2005tauri Micromonas EEH58637 Petrie et al., 2010a pusilla (SEQ ID NO: 7)Δ5-desaturase Algae Mantoniella CAQ30478 482 Hoffmann et al., 2008squamata Plant Anemone N/A Sayanova et al., 2007 leveillei ω3-desaturaseFungi Pythium FW362186.1 359 Xue et al., 2012; aphanidermatumWO2008/054565 Fungi Phytophthora FW362214.1 363 Xue et al., 2012;(oomycete) sojae WO2008/054565 Fungi Phytophthora FW362213.1 361 Xue etal., 2012; (oomycete) ramorum WO2008/054565

In an embodiment the Δ6-desaturase has greater activity on an ω3substrate than the corresponding ω6 substrate and has activity on ALA toproduce octadecatetraenoic acid (stearidonic acid, SDA, 18:4Δ6,9,12, 15,ω3) with an efficiency of at least 30%, more preferably at least 40%, ormost preferably at least 50% when expressed from an exogenouspolynucleotide in a recombinant cell such as a plant cell, or at least35% when expressed in a yeast cell. In one embodiment, the Δ6-desaturasehas greater activity, for example, at least about a 2-fold greaterΔ6-desaturase activity, on ALA than LA as fatty acid substrate. Inanother embodiment, the Δ6-desaturase has greater activity, for example,at least about 5 fold greater Δ6-desaturase activity or at least 10-foldgreater activity, on ALA-CoA as fatty acid substrate than on ALA joinedto the sn-2 position of PC as fatty acid substrate. In a furtherembodiment, the Δ6-desaturase has activity on both fatty acid substratesALA-CoA and on ALA joined to the sn-2 position of PC.

In one embodiment, the Δ6-desaturase has no detectable Δ5-desaturaseactivity on ETA. In another embodiment, the Δ6-desaturase comprisesamino acids having a sequence as provided in SEQ ID NO:9, SEQ ID NO:12or SEQ ID NO:13, a biologically active fragment thereof, or an aminoacid sequence which is at least 77% identical to SEQ ID NO:9, SEQ IDNO:12 or SEQ ID NO:13. In another embodiment, the Δ6-desaturasecomprises amino acids having a sequence as provided in SEQ ID NO:12 orSEQ ID NO:13, a biologically active fragment thereof, or an amino acidsequence which is at least 67% identical to one or both of SEQ ID NO:12or SEQ ID NO:13. The Δ6-desaturase may also have Δ8-desaturase activity.

As used herein, a “Δ8-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the8^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. The Δ8-desaturase is at least capable of converting ETrA toETA. Preferably, the Δ8-desaturase is capable of converting ETrA-CoA toETA-CoA, i.e. it is an acyl-CoA desaturase. In an embodiment, theΔ8-desaturase is capable of converting ETrA esterified at the sn-2position of PC. Preferably the Δ8-desaturase has greater activity onETrA-CoA than on ETrA-PC. The Δ8-desaturase may also have activity as aΔ6-desaturase, being termed a Δ6/Δ8 bifunctional desaturase, so long asit has greater Δ8-desaturase activity on ETrA than Δ6-desaturaseactivity on ALA. Examples of Δ8-desaturases are listed in Table 1. Inone embodiment, the Δ8-desaturase comprises amino acids having asequence as provided in SEQ ID NO:37, a biologically active fragmentthereof, or an amino acid sequence which is at least 80% identical toSEQ ID NO:37.

As used herein, an “ω3-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the3rd carbon-carbon bond from the methyl end of a fatty acid substrate. Aω3-desaturase therefore may convert LA to ALA and GLA to SDA (all C18fatty acids), or DGLA to ETA and/or ARA to EPA (C20 fatty acids). Someω3-desaturases (group I) have activity only on C18 substrates, such asplant and cyanobacterial ω3-desaturases. Such ω3-desaturases are alsoΔ15-desaturases. Other ω3-desaturases have activity on C20 substrateswith no activity (group II) or some activity (group III) on C18substrates. Such ω3-desaturases are also Δ17-desaturases. Preferredω3-desaturases are group III type which convert LA to ALA, GLA to SDA,DGLA to ETA and ARA to EPA, such as the Pichia pastoris ω3-desaturase(SEQ ID NO: 6). Examples of ω3-desaturases include those described byPereira et al. (2004a) (Saprolegnia diclina ω3-desaturase, group H),Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al.(1997) (C. elegans ω3-desaturase, group III). In a preferred embodiment,the ω3-desaturase is a fungal ω3-desaturase. As used herein, a “fungalω3-desaturase” refers to an ω3-desaturase which is from a fungal source,including an oomycete source, or a variant thereof whose amino acidsequence is at least 95% identical thereto. Genes encoding numerousω3-desaturases have been isolated from fungal sources such as, forexample, from Phytophthora infestans (Accession No. CAJ30870,WO2005083053; SEQ ID NO: 47), Saprolegnia diclina (Accession No.AAR20444, Pereira et al., 2004a & U.S. Pat. No. 7,211,656), Pythiumirregulare (WO2008022963, Group II; SEQ ID NO: 49), Mortierella alpina(Sakuradani et al., 2005; Accession No. BAD91495; WO2006019192),Thalassiosira pseudonana (Armbrust et al., 2004; Accession No.XP_002291057; WO2005012316, SEQ ID NO: 48), Lachancea kluyveri (alsoknown as Saccharomyces kluyveri; Oura et al., 2004; Accession No.AB118663). Xue et al. (2012) describes ω3-desaturases from the oomycetesPythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorumwhich were able to efficiently convert ω6 fatty acid substrates to thecorresponding ω3 fatty acids, with a preference for C20 substrates, i.e.they had stronger 017-desaturase activity than Δ15-desaturase activity.These enzymes lacked Δ12-desaturase activity, but could use fatty acidsin both acyl-CoA and phospholipid fraction as substrates.

In a more preferred embodiment, the fungal ω3-desaturase is the Pichiapastoris (also known as Komagataella pastoris)ω3-desaturase/Δ15-desaturase (Zhang et al., 2008; Accession No.EF116884; SEQ ID NO: 6), or a polypeptide which is at least 95%identical thereto.

In an embodiment, the ω3-desaturase is at least capable of convertingone of ARA to EPA, DGLA to ETA, GLA to SDA, both ARA to EPA and DGLA toETA, both ARA to EPA and GLA to SDA, or all three of these.

In one embodiment, the ω3-desaturase has Δ17-desaturase activity on aC20 fatty acid which has at least three carbon-carbon double bonds,preferably ARA. In another embodiment, the ω3-desaturase hasΔ15-desaturase activity on a C18 fatty acid which has threecarbon-carbon double bonds, preferably GLA. Preferably, both activitiesare present.

As used herein, a “Δ12-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the12^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Δ12-desaturases typically convert eitheroleoyl-phosphatidylcholine or oleoyl-CoA tolinoleoyl-phosphatidylcholine (18:1-PC) or linoleoyl-CoA (18:1-CoA),respectively. The subclass using the PC linked substrate are referred toas phospholipid-dependent Δ12-desaturases, the latter sublclass asacyl-CoA dependent Δ12-desaturases. Plant and fungal Δ12-desaturases aregenerally of the former sub-class, whereas animal Δ12-desaturases are ofthe latter subclass, for example the Δ12-desaturases encoded by genescloned from insects by Thou et al. (2008). Many other Δ12-desaturasesequences can be easily identified by searching sequence databases.

As used herein, a “Δ15-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the15^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. Numerous genes encoding Δ15-desaturases have been cloned fromplant and fungal species. For example, U.S. Pat. No. 5,952,544 describesnucleic acids encoding plant Δ15-desaturases (FAD3). These enzymescomprise amino acid motifs that were characteristic of plantΔ15-desaturases. WO200114538 describes a gene encoding soybean FAD3.Many other Δ15-desaturase sequences can be easily identified bysearching sequence databases.

As used herein, a “Δ17-desaturase” refers to a protein which performs adesaturase reaction that introduces a carbon-carbon double bond at the17^(th) carbon-carbon bond from the carboxyl end of a fatty acidsubstrate. A Δ17-desaturase is also regarded as an ω3-desaturase if itacts on a C20 substrate to introduce a desaturation at the ω3 bond.

In a preferred embodiment, the Δ12-desaturase and/or Δ15-desaturase is afungal Δ12-desaturase or fungal Δ15-desaturase. As used herein, a“fungal Δ12-desaturase” or “a fungal Δ15-desaturase” refers to aΔ12-desaturase or Δ15-desaturase which is from a fungal source,including an oomycete source, or a variant thereof whose amino acidsequence is at least 95% identical thereto. Genes encoding numerousdesaturases have been isolated from fungal sources. U.S. Pat. No.7,211,656 describes a Δ12 desaturase from Saprolegnia diclina.WO2009016202 describes fungal desaturases from Helobdella robusta,Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosigabrevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola,Naegleria gruben, Nectria haematoeocca, Nematostella vectensis,Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens,Postia placenta, Selaginella moellendorffii and Microdochium nivale.WO2005/012316 describes a Δ12-desaturase from Thalassiosira pseudonanaand other fungi. WO2003/099216 describes genes encoding fungalΔ12-desaturases and Δ15-desaturases isolated from Neurospora crassa,Aspergillus nidulans, Botrytis cinerea and Mortierella alpina.WO2007133425 describes fungal Δ15 desaturases isolated from:Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans,Neurospora crassa, Fusarium graminearum, Fusarium moniliforme andMagnaporthe grisea. A preferred Δ12 desaturase is from Phytophthorasojae (Ruiz-Lopez et al., 2012).

A distinct subclass of fungal Δ12-desaturases, and of fungalΔ15-desaturases, are the bifunctional fungal Δ12/Δ15-desaturases. Genesencoding these have been cloned from Fusarium monoliforme (Accession No.DQ272516, Damude et al., 2006), Acanthamoeba castellanii (Accession No.EF017656, Sayanova et al., 2006), Perkinsus marinus (WO2007042510),Claviceps purpurea (Accession No. EF536898, Meesapyodsuk et al., 2007)and Coprinus cinereus (Accession No. AF269266, Zhang et al., 2007).

In another embodiment, the ω3-desaturase has at least some activity on,preferably greater activity on, an acyl-CoA substrate than acorresponding acyl-PC substrate. As used herein, a “correspondingacyl-PC substrate” refers to the fatty acid esterified at the sn-2position of phosphatidylcholine (PC) where the fatty acid is the samefatty acid as in the acyl-CoA substrate. For example, the acyl-CoAsubstrate may be ARA-CoA and the corresponding acyl-PC substrate is sn-2ARA-PC. In an embodiment, the activity is at least two-fold greater.Preferably, the ω3-desaturase has at least some activity on both anacyl-CoA substrate and its corresponding acyl-PC substrate and hasactivity on both C18 and C20 substrates. Examples of such ω3-desaturasesare known amongst the cloned fungal desaturases listed above.

In a further embodiment, the ω3-desaturase comprises amino acids havinga sequence as provided in SEQ ID NO:6, a biologically active fragmentthereof, or an amino acid sequence which is at least 60% identical toSEQ ID NO:6, preferably at least 90% or at least 95% identical to SEQ IDNO:6.

In yet a further embodiment, a desaturase for use in the presentinvention has greater activity on an acyl-CoA substrate than acorresponding acyl-PC substrate. In another embodiment, a desaturase foruse in the present invention has greater activity on an acyl-PCsubstrate than a corresponding acyl-CoA substrate, but has some activityon both substrates. As outlined above, a “corresponding acyl-PCsubstrate” refers to the fatty acid esterified at the sn-2 position ofphosphatidylcholine (PC) where the fatty acid is the same fatty acid asin the acyl-CoA substrate. In an embodiment, the greater activity is atleast two-fold greater. In an embodiment, the desaturase is a Δ5 orΔ6-desaturase, or an ω3-desaturase, examples of which are provided, butnot limited to, those listed in Table 2. To test which substrate adesaturase acts on, namely an acyl-CoA or an acyl-PC substrate, assayscan be carried out in yeast cells as described in Domergue et al. (2003and 2005). Acyl-CoA substrate capability for a desaturase can also beinferred when an elongase, when expressed together with the desturase,has an enzymatic conversion efficiency in plant cells of at least about90% where the elongase catalyses the elongation of the product of thedesaturase. On this basis, the Δ5-desaturase and Δ4-desaturasesexpressed from the GA7 construct (Examples 2 and 3) and variants thereof(Example 4) are capable of desaturating their respective acyl-CoAsubstrates, ETA-CoA and DPA-CoA.

Elongases

Biochemical evidence suggests that the fatty acid elongation consists of4 steps: condensation, reduction, dehydration and a second reduction. Inthe context of this invention, an “elongase” refers to the polypeptidethat catalyses the condensing step in the presence of the other membersof the elongation complex, under suitable physiological conditions. Ithas been shown that heterologous or homologous expression in a cell ofonly the condensing component (“elongase”) of the elongation proteincomplex is required for the elongation of the respective acyl chain.Thus, the introduced elongase is able to successfully recruit thereduction and dehydration activities from the transgenic host to carryout successful acyl elongations. The specificity of the elongationreaction with respect to chain length and the degree of desaturation offatty acid substrates is thought to reside in the condensing component.This component is also thought to be rate limiting in the elongationreaction.

As used herein, a “Δ5-elongase” is at least capable of converting EPA toDPA. Examples of Δ5-elongases include those disclosed in WO2005/103253.In one embodiment, the Δ5-elongase has activity on EPA to produce DPAwith an efficiency of at least 60%, more preferably at least 65%, morepreferably at least 70% or most preferably at least 80% or 90%. In afurther embodiment, the Δ5-elongase comprises an amino acid sequence asprovided in SEQ ID NO:25, a biologically active fragment thereof, or anamino acid sequence which is at least 47% identical to SEQ ID NO:25. Ina further embodiment, the Δ6-elongase is from Ostreococcus taurii orOstreococcus lucimarinus (US2010/088776).

As used herein, a “Δ6-elongase” is at least capable of converting SDA toETA. Examples of Δ6-elongases include those listed in Table 1. In oneembodiment, the elongase comprises amino acids having a sequence asprovided in SEQ ID NO:16, a biologically active fragment thereof (suchas the fragment provided as SEQ ID NO:17), or an amino acid sequencewhich is at least 55% identical to one or both of SEQ ID NO:16 or SEQ IDNO:17. In an embodiment, the Δ6-elongase is from Physcomitrella patens(Zank et al., 2002; Accession No. AF428243) or Thalassiosira pseudonana(Ruiz-Lopez et al., 2012).

As used herein, a “Δ9-elongase” is at least capable of converting ALA toETrA. Examples of Δ9-elongases include those listed in Table 1. In oneembodiment, the Δ9-elongase comprises amino acids having a sequence asprovided in SEQ ID NO:29, a biologically active fragment thereof, or anamino acid sequence which is at least 80% identical to SEQ ID NO:29. Inanother embodiment, the Δ9-elongase comprises amino acids having asequence as provided in SEQ ID NO:31, a biologically active fragmentthereof, or an amino acid sequence which is at least 81% identical toSEQ ID NO:31. In another embodiment, the Δ9-elongase comprises aminoacids having a sequence as provided in SEQ ID NO:33, a biologicallyactive fragment thereof, or an amino acid sequence which is at least 50%identical to SEQ ID NO:33. In another embodiment, the Δ9-elongasecomprises amino acids having a sequence as provided in SEQ ID NO:35, abiologically active fragment thereof, or an amino acid sequence which isat least 50% identical to SEQ ID NO:35. In a further embodiment, theΔ9-elongase has greater activity on an ω6 substrate than thecorresponding ω3 substrate, or the converse.

As used herein, the term “has greater activity on an ω6 substrate thanthe corresponding ω3 substrate” refers to the relative activity of theenzyme on substrates that differ by the action of an ω3 desaturase.Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

An elongase with Δ6-elongase and Δ9-elongase activity is at leastcapable of (i) converting SDA to ETA and (ii) converting ALA to ETrA andhas greater Δ6-elongase activity than Δ9-elongase activity. In oneembodiment, the elongase has an efficiency of conversion on SDA toproduce ETA which is at least 50%, more preferably at least 60%, and/oran efficiency of conversion on ALA to produce ETrA which is at least 6%or more preferably at least 9%. In another embodiment, the elongase hasat least about 6.5 fold greater Δ6-elongase activity than Δ9-elongaseactivity. In a further embodiment, the elongase has no detectableΔ5-elongase activity.

Other Enzymes

The transgenes introduced into the recombinant cell such as a microbialcell, or transgenic plant or part thereof may also encode an LPAAT. Asused herein, the term “1-acyl-glycerol-3-phosphate acyltransferase”(LPAAT), also termed lysophosphatidic acid-acyltransferase oracylCoA-lysophosphatidate-acyltransferase, refers to a protein whichacylates sn-1-acyl-glycerol-3-phosphate (sn-1 G-3-P) at the sn-2position to form phosphatidic acid (PA). Thus, the term“1-acyl-glycerol-3-phosphate acyltransferase activity” refers to theacylation of (sn-1 G-3-P) at the sn-2 position to produce PA (EC2.3.1.51). Preferred LPAATs are those that can use a polyunsaturated C22acyl-CoA as substrate to transfer the polyunsaturated C22 acyl group tothe sn-2 position of LPA, forming PA. In an embodiment, thepolyunsaturated C22 acyl-CoA is DHA-CoA and/or DPA-CoA. Such LPAATs areexemplified in Example 7 and can be tested as described therein. In anembodiment, an LPAAT useful for the invention comprises amino acidshaving a sequence as provided in any one of SEQ ID NOs: 40 to 46, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to any one or more of SEQ ID NOs: 40 to 46. Inanother embodiment, the LPAAT does not have amino acids having asequence as provided in any one of SEQ ID NO: 44. In a preferredembodiment, an LPAAT useful for the invention which can use a C22polyunsaturated fatty acyl-CoA substrate, preferably DHA-CoA and/orDPA-CoA, comprises amino acids having a sequence as provided in any oneof SEQ ID NOs: 41, 42 and 44, a biologically active fragment thereof, oran amino acid sequence which is at least 40% identical to any one ormore of SEQ ID NOs: 41, 42 and 44. In a preferred embodiment, an LPAATuseful for the invention which can use a C22 polyunsaturated fattyacyl-CoA substrate, preferably DHA-CoA and/or DPA-CoA, comprises aminoacids having a sequence as provided in any one of SEQ ID NOs: 41 or 42,a biologically active fragment thereof, or an amino acid sequence whichis at least 40% identical to any one or both of SEQ ID NOs: 41 and 42.In an embodiment in which the genetic construct expresses aΔ4-desaturase in the transgenic cell and/or the transgenic cell producesDHA, the LPAAT is preferably an LPAAT other the Mortierella alpina LPAATwhose amino acid sequence is set forth as SEQ ID NO: 44. Alternatively,if the genetic construct does not express a Δ4-desaturase in thetransgenic cell and/or the transgenic cell produces DPA but not DHA, theLPAAT is preferably the Mortierella alpina LPAAT whose amino acidsequence is set forth as SEQ ID NO: 44 or another LPAAT which is capableof using DPA-CoA as a substrate to transfer the DPA to LPA, forming DAGhaving DPA at the sn-2 position.

The transgenes introduced into the recombinant cell, transgenic plant orpart thereof may also encode a DGAT. As used herein, the term“diacylglycerol acyltransferase” (EC 2.3.1.20; DGAT) refers to a proteinwhich transfers a fatty acyl group from acyl-CoA to a diacylglycerolsubstrate to produce a triacylglycerol. Thus, the term “diacylglycerolacyltransferase activity” refers to the transfer of acyl-CoA todiacylglycerol to produce triacylglycerol. There are three known typesof DGAT referred to as DGAT1, DGAT2 and DGAT3 respectively. DGAT1polypeptides typically have 10 transmembrane domains, DGAT2 typicallyhave 2 transmembrane domains, whilst DGAT3 is typically soluble.Examples of DGAT1 polypeptides include polypeptides encoded by DGAT1genes from Aspergillus fumigatus (Accession No. XP_755172), Arabidopsisthaliana (CAB44774), Ricinus communis (AAR11479), Vernicia fordii(ABC94472), Vernonia galamensis (ABV21945, ABV21946), Euonymus alatus(AAV31083), Caenorhabditis elegans (AAF82410), Rattus norvegicus(NP_445889), Homo sapiens (NP_036211), as well as variants and/ormutants thereof. Examples of DGAT2 polypeptides include polypeptidesencoded by DGAT2 genes from Arabidopsis thaliana (Accession No.NP_566952), Ricinus communis (AAY16324), Vernicia fordii (ABC94474),Mortierella ramanniana (AAK84179), Homo sapiens (Q96PD7, Q58HT5), Bostaurus (Q70VD8), Mus musculus (AAK84175), Micromonas CCMP1545, as wellas variants and/or mutants thereof. Examples of DGAT3 polypeptidesinclude polypeptides encoded by DGAT3 genes from peanut (Arachishypogaea, Saha, et al., 2006), as well as variants and/or mutantsthereof.

Polypeptides/Peptides

The terms “polypeptide” and “protein” are generally usedinterchangeably.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The 25% identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 15 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 15 amino acids. More preferably,the query sequence is at least 50 amino acids in length, and the GAPanalysis aligns the two sequences over a region of at least 50 aminoacids. More preferably, the query sequence is at least 100 amino acidsin length and the GAP analysis aligns the two sequences over a region ofat least 100 amino acids. Even more preferably, the query sequence is atleast 250 amino acids in length and the GAP analysis aligns the twosequences over a region of at least 250 amino acids. Even morepreferably, the GAP analysis aligns two sequences over their entirelength. The polypeptide or class of polypeptides may have the sameenzymatic activity as, or a different activity than, or lack theactivity of, the reference polypeptide. Preferably, the polypeptide hasan enzymatic activity of at least 10%, at least 50%, at least 75% or atleast 90%, of the activity of the reference polypeptide.

As used herein a “biologically active” fragment is a portion of apolypeptide defined herein which maintains a defined activity of afull-length reference polypeptide, for example possessing desaturaseand/or elongase activity or other enzyme activity. Biologically activefragments as used herein exclude the full-length polypeptide.Biologically active fragments can be any size portion as long as theymaintain the defined activity. Preferably, the biologically activefragment maintains at least 10%, at least 50%, at least 75% or at least90%, of the activity of the full length protein.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 76%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ 11.) NO.

Amino acid sequence variants/mutants of the polypeptides of the definedherein can be prepared by introducing appropriate nucleotide changesinto a nucleic acid defined herein, or by in vitro synthesis of thedesired polypeptide. Such variants/mutants include, for example,deletions, insertions or substitutions of residues within the amino acidsequence. A combination of deletion, insertion and substitution can bemade to arrive at the final construct, provided that the final peptideproduct possesses the desired enzyme activity.

Mutant (altered) peptides can be prepared using any technique known inthe art. For example, a polynucleotide defined herein can be subjectedto in vitro mutagenesis or DNA shuffling techniques as broadly describedby Harayama (1998). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess, for example, desaturase or elongase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites which are not conserved amongst naturally occurringdesaturases or elongases. These sites are preferably substituted in arelatively conservative manner in order to maintain enzyme activity.Such conservative substitutions are shown in Table 3 under the headingof “exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 3. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell.

Polynucleotides

The invention also provides for the use of polynucleotides which may be,for example, a gene, an isolated polynucleotide, a chimeric geneticconstruct such as a T-DNA molecule, or a chimeric DNA. It may be DNA orRNA of genomic or synthetic origin, double-stranded or single-stranded,and combined with carbohydrate, lipids, protein or other materials toperform a particular activity defined herein. The term “polynucleotide”is used interchangeably herein with the term “nucleic acid molecule”.

In an embodiment, the polynucleotide is non-naturally occurring.Examples of non-naturally occurring polynucleotides include, but are notlimited to, those that have been mutated (such as by using methodsdescribed herein), and polynucleotides where an open reading frameencoding a protein is operably linked to a promoter to which it is notnaturally associated (such as in the constructs described herein).

TABLE 3 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals in which case thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (hnRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the messenger RNA (mRNA) transcript. The mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide. The term “gene” includes a synthetic or fusion moleculeencoding all or part of the proteins described herein and acomplementary nucleotide sequence to any one of the above.

As used herein, a “chimeric DNA” or “chimeric genetic construct” orsimilar refers to any DNA molecule that is not a native DNA molecule inits native location, also referred to herein as a “DNA construct”.Typically, a chimeric DNA or chimeric gene comprises regulatory andtranscribed or protein coding sequences that are not found operablylinked together in nature i.e. that are heterologous with respect toeach other. Accordingly, a chimeric DNA or chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature.

An “endogenous gene” refers to a native gene in its natural location inthe genome of an organism. As used herein, “recombinant nucleic acidmolecule”, “recombinant polynucleotide” or variations thereof refer to anucleic acid molecule which has been constructed or modified byrecombinant DNA technology. The terms “foreign polynucleotide” or“exogenous polynucleotide” or “heterologous polynucleotide” and the likerefer to any nucleic acid which is introduced into the genome of a cellby experimental manipulations. Foreign or exogenous genes may be genesthat are inserted into a non-native organism, native genes introducedinto a new location within the native host, or chimeric genes. A“transgene” is a gene that has been introduced into the genome by atransformation procedure. The terms “genetically modified”, “transgenic”and variations thereof include introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny. A “genomic region” as usedherein refers to a position within the genome where a transgene, orgroup of transgenes (also referred to herein as a cluster), have beeninserted into a cell, or an ancestor thereof. Such regions only comprisenucleotides that have been incorporated by the intervention of man suchas by methods described herein.

The term “exogenous” in the context of a polynucleotide refers to thepolynucleotide when present in a cell in an altered amount compared toits native state. In one embodiment, the cell is a cell that does notnaturally comprise the polynucleotide. However, the cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide. An exogenouspolynucleotide includes polynucleotides which have not been separatedfrom other components of the transgenic (recombinant) cell, or cell-freeexpression system, in which it is present, and polynucleotides producedin such cells or cell-free systems which are subsequently purified awayfrom at least some other components. The exogenous polynucleotide(nucleic acid) can be a contiguous stretch of nucleotides existing innature, or comprise two or more contiguous stretches of nucleotides fromdifferent sources (naturally occurring and/or synthetic) joined to forma single polynucleotide. Typically such chimeric polynucleotidescomprise at least an open reading frame encoding a polypeptide operablylinked to a promoter suitable of driving transcription of the openreading frame in a cell of interest.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, more preferably at least 93%, more preferably at least 94%, morepreferably at least 95%, more preferably at least 96%, more preferablyat least 97%, more preferably at least 98%, more preferably at least99%, more preferably at least 99.1%, more preferably at least 99.2%,more preferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Polynucleotides may possess, when compared to naturally occurringmolecules, one or more mutations which are deletions, insertions, orsubstitutions of nucleotide residues. Polynucleotides which havemutations relative to a reference sequence can be either naturallyoccurring (that is to say, isolated from a natural source) or synthetic(for example, by performing site-directed mutagenesis or DNA shufflingon the nucleic acid as described above). It is thus apparent thatpolynucleotides can be either from a naturally occurring source orrecombinant. Preferred polynucleotides are those which have codingregions that are codon-optimised for translation in plant cells, as isknown in the art.

Recombinant Vectors

Recombinant expression can be used to produce recombinant cells, orplants or plant parts of the invention. Recombinant vectors containheterologous polynucleotide sequences, that is, polynucleotide sequencesthat are not naturally found adjacent to polynucleotide moleculesdefined herein that preferably are derived from a species other than thespecies from which the polynucleotide molecule(s) are derived. Thevector can be either RNA or DNA and typically is a plasmid. Plasmidvectors typically include additional nucleic acid sequences that providefor easy selection, amplification, and transformation of the expressioncassette in prokaryotic cells, e.g., pUC-derived vectors, pSK-derivedvectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors,or preferably binary vectors containing one or more T-DNA regions.Additional nucleic acid sequences include origins of replication toprovide for autonomous replication of the vector, selectable markergenes, preferably encoding antibiotic or herbicide resistance, uniquemultiple cloning sites providing for multiple sites to insert nucleicacid sequences or genes encoded in the nucleic acid construct, andsequences that enhance transformation of prokaryotic and eukaryotic(especially plant) cells. The recombinant vector may comprise more thanone polynucleotide defined herein, for example three, four, five or sixpolynucleotides defined herein in combination, preferably a chimericgenetic construct described herein, each polynucleotide being operablylinked to expression control sequences that are operable in the cell ofinterest. Preferably the expression control sequences include, or areall, heterologous promoters i.e. are heterologous with respect to thecoding regions they control. More than one polynucleotide definedherein, for example 3, 4, 5 or 6 polynucleotides, preferably 7 or 8polynucleotides each encoding a different polypeptide, are preferablycovalently joined together in a single recombinant vector, preferablywithin a single T-DNA molecule, which may then be introduced as a singlemolecule into a cell to form a recombinant cell according to theinvention, and preferably integrated into the genome of the recombinantcell, for example in a transgenic plant. The integration into the genomemay be into the nuclear genome or into a plastid genome in thetransgenic plant. Thereby, the polynucleotides which are so joined willbe inherited together as a single genetic locus in progeny of therecombinant cell or plant. The recombinant vector or plant may comprisetwo or more such recombinant vectors, each containing multiplepolynucleotides, for example wherein each recombinant vector comprises3, 4, 5 or 6 polynucleotides.

“Operably linked” as used herein refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence, such as a polynucleotide definedherein, if it stimulates or modulates the transcription of the codingsequence in an appropriate cell. Generally, promoter transcriptionalregulatory elements that are operably linked to a transcribed sequenceare physically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

When there are multiple promoters present, each promoter mayindependently be the same or different. Preferably, at least 3 and up toa maximum of 6 different promoter sequences are used in the recombinantvector to control expression of the exogenous polynucleotides.

Recombinant molecules such as the chimeric DNAs or genetic constructsmay also contain (a) one or more secretory signals which encode signalpeptide sequences, to enable an expressed polypeptide defined herein tobe secreted from the cell that produces the polypeptide or which providefor localisation of the expressed polypeptide, for example for retentionof the polypeptide in the endoplasmic reticulum (ER) in the cell ortransfer into a plastid, and/or (b) contain fusion sequences which leadto the expression of nucleic acid molecules as fusion proteins. Examplesof suitable signal segments include any signal segment capable ofdirecting the secretion or localisation of a polypeptide defined herein.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules defined herein.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. By “markergene” is meant a gene that imparts a distinct phenotype to cellsexpressing the marker gene and thus allows such transformed cells to bedistinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked. The actual choice of a marker is notcrucial as long as it is functional (i.e., selective) in combinationwith the cells of choice such as a plant cell.

Examples of selectable markers are markers that confer antibioticresistance such as ampicillin, erythromycin, chloramphenicol ortetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphatesynthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as,for example, described by Hinchee et al. (1988), or preferably a bargene conferring resistance against bialaphos as, for example, describedin WO91/02071.

Preferably, the nucleic acid construct is stably incorporated into thegenome of the cell, such as the plant cell. Accordingly, the nucleicacid may comprise appropriate elements which allow the molecule to beincorporated into the genome, preferably the right and left bordersequences of a T-DNA molecule, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of thecell.

Expression

As used herein, an expression vector is a DNA vector that is capable oftransforming a host cell and of effecting expression of one or morespecified polynucleotide molecule(s). Expression vectors of the presentinvention can direct gene expression in plant cells or in recombinantcells such as microbial cells. Expression vectors useful for theinvention contain regulatory sequences such as transcription controlsequences, translation control sequences, origins of replication, andother regulatory sequences that are compatible with the recombinant celland that control the expression of polynucleotide molecules of thepresent invention. In particular, polynucleotides or vectors useful forthe present invention include transcription control sequences.Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter and enhancer sequences.Suitable transcription control sequences include any transcriptioncontrol sequence that can function in at least one of the recombinantcells of the present invention. The choice of the regulatory sequencesused depends on the target organism such as a plant and/or target organor tissue of interest. Such regulatory sequences may be obtained fromany eukaryotic organism such as plants or plant viruses, or may bechemically synthesized. A variety of such transcription controlsequences are known to those skilled in the art. Particularly preferredtranscription control sequences are promoters active in directingtranscription in plants, either constitutively or stage and/or tissuespecific, depending on the use of the plant or parts thereof.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, and the light-induciblepromoter from the small subunit of the ribulose-1,5-bis-phosphatecarboxylase.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. Many examples are well known in the art. Avariety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of genes in plant cells, or it may also beadvantageous to employ organ-specific promoters.

As used herein, the term “seed specific promoter” or variations thereofrefer to a promoter that preferentially, when compared to other planttissues, directs gene transcription in a developing seed of a plant,preferably a Brassica sp., Camelina sativa or G. max plant. In anembodiment, the seed specific promoter is expressed at least 5-fold morestrongly in the developing seed of the plant relative to the leavesand/or stems of the plant, and is preferably expressed more strongly inthe embryo of the developing seed compared to other plant tissues.Preferably, the promoter only directs expression of a gene of interestin the developing seed, and/or expression of the gene of interest inother parts of the plant such as leaves is not detectable by Northernblot analysis and/or RT-PCR. Typically, the promoter drives expressionof genes during growth and development of the seed, in particular duringthe phase of synthesis and accumulation of storage compounds in theseed. Such promoters may drive gene expression in the entire plantstorage organ or only part thereof such as the seedcoat, orcotyledon(s), preferably in the embryos, in seeds of dicotyledonousplants or the endosperm or aleurone layer of a seeds of monocotyledonousplants.

Preferred promoters for seed-specific expression include i) promotersfrom genes encoding enzymes involved in fatty acid biosynthesis andaccumulation in seeds, such as fatty acid desaturases and elongases, ii)promoters from genes encoding seed storage proteins, and iii) promotersfrom genes encoding enzymes involved in carbohydrate biosynthesis andaccumulation in seeds. Seed specific promoters which are suitable arethe oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), theVicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosinpromoter (WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S.Pat. No. 5,504,200), the Brassica Bce4 promoter (WO91/13980) or thelegumin LeB4 promoter from Vicia faba (Baumlein et al., 1992), andpromoters which lead to the seed-specific expression in monocots such asmaize, barley, wheat, rye, rice and the like. Notable promoters whichare suitable are the barley lpt2 or lpt1 gene promoter (WO95/15389 andWO95/23230) or the promoters described in WO99/16890 (promoters from thebarley hordein gene, the rice glutelin gene, the rice oryzin gene, therice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, themaize zein gene, the oat glutelin gene, the sorghum kasirin gene, therye secalin gene). Other promoters include those described by Broun etal. (1998), Potenza et al. (2004), US20070192902 and US20030159173. Inan embodiment, the seed specific promoter is preferentially expressed indefined parts of the seed such as the embryo, cotyledon(s) or theendosperm. Examples of such specific promoters include, but are notlimited to, the FP1 promoter (Ellerstrom et al., 1996), the pea leguminpromoter (Perrin et al., 2000), the bean phytohemagglutnin promoter(Perrin et al., 2000), the conlinin 1 and conlinin 2 promoters for thegenes encoding the flax 2S storage proteins (Cheng et al., 2010), thepromoter of the FAE1 gene from Arabidopsis thaliana, the BnGLP promoterof the globulin-like protein gene of Brassica napus, the LPXR promoterof the peroxiredoxin gene from Linum usitatissimum.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of the polynucleotideof the present invention, or preferably is heterologous with respect tothe coding region of the enzyme to be produced, and can be specificallymodified if desired so as to increase translation of mRNA. For a reviewof optimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. Nos. 5,362,865 and5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the chimeric vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in plant cells. The nopaline synthase3′ untranslated region, the 3′ untranslated region from pea smallsubunit Rubisco gene, the 3′ untranslated region from soybean 7S seedstorage protein gene or a flax conlinin gene are commonly used in thiscapacity. The 3′ transcribed, non-translated regions containing thepolyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genesare also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide molecule by manipulating, for example, thenumber of copies of the polynucleotide molecule within a host cell, theefficiency with which those polynucleotide molecules are transcribed,the efficiency with which the resultant transcripts are translated, andthe efficiency of post-translational modifications. Recombinanttechniques useful for increasing the expression of polynucleotidemolecules defined herein include, but are not limited to, integration ofthe polynucleotide molecule into one or more host cell chromosomes,addition of stability sequences to mRNAs, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of polynucleotide molecules to correspond to the codonusage of the host cell, and the deletion of sequences that destabilizetranscripts.

Transgenic Plants

The term “plant” as used herein as a noun refers to whole plants, but asused as an adjective refers to any substance which is present in,obtained from, derived from, or related to a plant, such as for example,plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g.pollen), seeds, plant cells and the like. The term “plant part” refersto all plant parts that comprise the plant DNA, including vegetativestructures such as, for example, leaves or stems, roots, floral organsor structures, pollen, seed, seed parts such as an embryo, endosperm,scutellum or seed coat, plant tissue such as, for example, vasculartissue, cells and progeny of the same, as long as the plant partsynthesizes lipid according to the invention.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a gene construct (“transgene”) not foundin a wild-type plant of the same species, variety or cultivar.Transgenic plants as defined in the context of the present inventioninclude plants and their progeny which have been genetically modifiedusing recombinant techniques to cause production of the lipid or atleast one polypeptide defined herein in the desired plant or plantorgan. Transgenic plant cells and transgenic plant parts havecorresponding meanings. A “transgene” as referred to herein has thenormal meaning in the art of biotechnology and includes a geneticsequence which has been produced or altered by recombinant DNA or RNAtechnology and which has been introduced into a plant cell. Thetransgene may include genetic sequences derived from a plant cell whichmay be of the same species, variety or cultivar as the plant cell intowhich the transgene is introduced or of a different species, variety orcultivar, or from a cell other than a plant cell. Typically, thetransgene has been introduced into the cell, such as a plant, by humanmanipulation such as, for example, by transformation but any method canbe used as one of skill in the art recognizes.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature grain orseed commonly has a moisture content of less than about 18-20%,preferably less than 10%. Brassica seed such as canola seed typicallyhas a moisture content of about 4-8% or 6-8% when mature, preferablybetween about 4% to about 6%. “Developing seed” as used herein refers toa seed prior to maturity, typically found in the reproductive structuresof the plant after fertilisation or anthesis, but can also refer to suchseeds prior to maturity which are isolated from a plant.

As used herein, the term “obtaining a plant part” or “obtaining a seed”refers to any means pf obtaining a plant part or seed, respectively,including harvesting of the plant parts or seed from plants in the fieldor in containment such as a glasshouse or growth chamber, or by purchaseor receipt from a supplier of the plant parts or seed. Standard growthconditions in a glasshouse include 22-24° C. daytime temperature and16-18° C. night-time temperature, with natural sunlight. The seed may besuitable for planting i.e. able to germinate and produce progeny plants,or alternatively has been processed in such a way that it is no longerable to germinate, e.g. cracked, polished or milled seed which is usefulfor food or feed applications, or for extraction of lipid of theinvention.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to storage energy in the form of, for example,proteins, carbohydrates, fatty acids and/or oils. Examples of plantstorage organs are seed, fruit, tuberous roots, and tubers. A preferredplant storage organ is seed.

The plants or plant parts of the invention or used in the invention arepreferably phenotypically normal. As used herein, the term“phenotypically normal” refers to a genetically modified plant or plantorgan, particularly a storage organ such as a seed, tuber or fruit nothaving a significantly reduced ability to grow and reproduce whencompared to an unmodified plant or plant organ. In an embodiment, thegenetically modified plant or plant organ which is phenotypically normalhas an ability to grow or reproduce which is essentially the same as anisogenic plant or organ not comprising the exogenous polynucleotide(s).Preferably, the biomass, growth rate, germination rate, storage organsize, pollen viability, male and female fertility, seed size and/or thenumber of viable seeds produced is not less than 90% of that of a plantlacking said exogenous polynucleotide when grown under identicalconditions. Preferably the pollen viability of the plant of theinvention, or plants produced from seed of the invention, is about 100%relative to the pollen viability of a corresponding wild-type plant.This term does not encompass features of the plant which may bedifferent to the wild-type plant but which do not affect the usefulnessof the plant for commercial purposes such as, for example, a ballerinaphenotype of seedling leaves.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetables orornamental plants. The plants of, or useful for, the invention may be:corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), mustard(Brassica juncea), flax (Linum usitatissimum), alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghumbicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritiumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumhirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta),coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananacomosus), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea(Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig(Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive(Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia intergrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), oats, or barley.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of oils from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseedplant may be other Brassicas, cotton, peanut, poppy, mustard, castorbean, sesame, sunflower, safflower, Camelina, Crambe or nut producingplants. The plant may produce high levels of oil in its fruit, such asolive, oil palm or coconut. Horticultural plants to which the presentinvention may be applied are lettuce, endive, or vegetable brassicasincluding cabbage, broccoli, or cauliflower. The present invention maybe applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.

In a further preferred embodiment, the non-transgenic plant used toproduce a transgenic plant of the invention produces oil, especially inthe seed, which has i) less than 20%, less than 10% or less than 5% 18:2fatty acids and/or ii) less than 10% or less than 5% 18:3 fatty acids.

In a preferred embodiment, the transgenic plant or part thereof ishomozygous for each and every gene (exogenous polynucleotide) that hasbeen introduced (transgene) so that its progeny do not segregate for thedesired phenotype. The transgenic plant may also be heterozygous for theintroduced transgene(s), preferably uniformly heterozygous for thetransgene, such as for example in F1 progeny which have been grown fromhybrid seed. Such plants may provide advantages such as hybrid vigour,well known in the art, or may be used in plant breeding or backcrossing.

Where relevant, the transgenic plant or part thereof may also compriseadditional transgenes encoding enzymes involved in the production ofLC-PUFAs such as, but not limited to, a Δ6-desaturase, a Δ9-elongase, aΔ8-desaturase, a Δ6-elongase, a Δ5-desaturase, an ω3-desaturase, aΔ4-desaturase, a Δ5-elongase, diacylglycerol acyltransferase, LPAAT, aΔ17-desaturase, a Δ15-desaturase and/or a Δ12 desaturase. Examples ofsuch enzymes with one of more of these activities are known in the artand include those described herein. In specific examples, the transgenicplant at least comprises a set of exogenous polynucleotides encoding;

a) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase anda Δ6-elongase,

b) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase anda Δ9-elongase,

c) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, aΔ6-elongase, and a Δ15-desaturase,

d) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, aΔ9-elongase, and a Δ15-desaturase,

e) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, aΔ6-elongase, and a Δ17-desaturase,

f) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, aΔ9-elongase, and a Δ17-desaturase,

g) an ω3-desaturase or a Δ15-desaturase, a Δ6-desaturase, aΔ5-desaturase, a Δ6-elongase and a Δ5-elongase,

h) an ω3-desaturase or a Δ15-desaturase, a Δ8-desaturase, aΔ5-desaturase, a Δ9-elongase and a Δ5-elongase,

i) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ6-desaturase, a Δ5-desaturase, a Δ6-elongase and an Δ5-elongase,

j) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ9-elongase and an Δ5-elongase,

k) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), anω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

l) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

m) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ6-elongase, anΔ5-elongase and optionally a Δ4-desaturase,

n) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a M2-desaturase, a ω3-desaturase and/or a Δ15-desaturase, a Δ6-desaturase,a Δ5-desaturase, a Δ6-elongase and an Δ5-elongase and optionally aΔ4-desaturase,

o) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), anω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, anΔ5-elongase and optionally a Δ4-desaturase,

p) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, aΔ5-elongase and optionally a Δ4-desaturase,

q) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ9-elongase, anΔ5-elongase and optionally a Δ4-desaturase, or

r) an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), aΔ12-desaturase, a ω3-desaturase and/or a Δ15-desaturase, aΔ8-desaturase, a Δ5-desaturase, a Δ9-elongase, an Δ5-elongase andoptionally a Δ4-desaturase.

In an embodiment, the exogenous polynucleotides encode set ofpolypeptides which are a Pythium irregulare Δ6-desaturase, aThraustochytrid Δ5-desaturase or an Emiliana huxleyi Δ5-desaturase, aPhyscomitrella patens Δ6-elongase, a Thraustochytrid Δ5-elongase or anOstreocccus taurii Δ5-elongase, a Phytophthora infestans ω3-desaturaseor a Pythium irregulare ω3-desaturase, and a ThraustochytridΔ4-desaturase.

In an embodiment, plants of, or used for, the invention are grown in thefield, preferably as a population of at least 1,000, 1,000,000 or2,000,000 plants that are essentially the same, or in an area of atleast 1 hectare or 2 hectares. Planting densities differ according tothe plant species, plant variety, climate, soil conditions, fertiliserrates and other factors as known in the art. For example, canola istypically grown at a planting density of 1.2-1.5 million plants perhectare. Plants are harvested as is known in the art, which may compriseswathing, windrowing and/or reaping of plants, followed by threshingand/or winnowing of the plant material to separate the seed from theremainder of the plant parts often in the form of chaff. Alternatively,seed may be harvested from plants in the field in a single process,namely combining.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in A. Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and P. Christou and H. Klee, Handbook of PlantBiotechnology, John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the exogenous nucleicacid molecules into the genome of the cell such that they aretransferred to progeny cells during cell division without the need forpositively selecting for their presence. Stable transformants, orprogeny thereof, can be selected by any means known in the art such asSouthern blots on chromosomal DNA or in situ hybridization of genomicDNA. Preferably, plant transformation is performed as described in theExamples herein.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues or plant organs or explants in tissueculture, for either transient expression or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863 or U.S. Pat. No. 5,159,135) including floral dipping methodsusing Agrobacterium or other bacteria that can transfer DNA into plantcells. The region of DNA to be transferred is defined by the bordersequences, and the intervening DNA (T-DNA) is usually inserted into theplant genome. Further, the integration of the T-DNA is a relativelyprecise process resulting in few rearrangements. In those plantvarieties where Agrobacterium-mediated transformation is efficient, itis the method of choice because of the facile and defined nature of thegene transfer. Preferred Agrobacterium transformation vectors arecapable of replication in E. coli as well as Agrobacterium, allowing forconvenient manipulations as described (Klee et al., In: Plant DNAInfectious Agents, Hohn and Schell, eds., Springer-Verlag, New York, pp.179-203 (1985).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO99/05265).

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contains a single genetic locus on one chromosome.Such transgenic plants can be referred to as being hemizygous for theadded gene(s). More preferred is a transgenic plant that is homozygousfor the added gene(s); i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by self-fertilisinga hemizygous transgenic plant, germinating some of the seed produced andanalyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Enhancing Exogenous RNA Levels and Stabilized Expression

Silencing Suppressors

In an embodiment, a plant cell, plant or plant part comprises anexogenous polynucleotide encoding a silencing suppressor protein.

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defense mechanism that can target both cellular andviral mRNAs for degradation PTGS occurs in plants or fungi stably ortransiently transformed with foreign (heterologous) or endogenous DNAand results in the reduced accumulation of RNA molecules with sequencesimilarity to the introduced nucleic acid.

It has widely been considered that co-expression of a silencingsuppressor with a transgene of interest will increase the levels of RNApresent in the cell transcribed from the transgene. Whilst this hasproven true for cells in vitro, significant side-effects have beenobserved in many whole plant co-expression studies. More specifically,as described in Mallory et al. (2002), Chapman et al. (2004), Chen etal. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al.(2007) and Meng et al. (2008) plants expressing silencing suppressors,generally under constitutive promoters, are often phenotypicallyabnormal to the extent that they are not useful for commercialproduction.

Recently, it has been found that RNA molecule levels can be increased,and/or RNA molecule levels stabilized over numerous generations, bylimiting the expression of the silencing suppressor to a seed of a plantor part thereof (WO2010/057246). As used herein, a “silencing suppressorprotein” or SSP is any polypeptide that can be expressed in a plant cellthat enhances the level of expression product from a different transgenein the plant cell, particularly over repeated generations from theinitially transformed plant. In an embodiment, the SSP is a viralsilencing suppressor or mutant thereof. A large number of viralsilencing suppressors are known in the art and include, but are notlimited to P19, V2, P38, Pe-Po and RPV-PO. In an embodiment, the viralsilencing suppressor comprises amino acids having a sequence as providedin SEQ ID NO:38, a biologically active fragment thereof, or an aminoacid sequence which is at least 50% identical to SEQ ID NO:38 and whichhas activity as a silencing suppressor.

As used herein, the terms “stabilising expression”, “stably expressed”,“stabilised expression” and variations thereof refer to level of the RNAmolecule being essentially the same or higher in progeny plants overrepeated generations, for example at least three, at least five or atleast 10 generations, when compared to isogenic plants lacking theexogenous polynucleotide encoding the silencing suppressor. However,this term(s) does not exclude the possibility that over repeatedgenerations there is some loss of levels of the RNA molecule whencompared to a previous generation, for example not less than a 10% lossper generation.

The suppressor can be selected from any source e.g. plant, viral, mammaletc. See WO2010/057246 for a list of viruses from which the suppressorcan be obtained and the protein (eg B2, P14 etc) or coding regiondesignation for the suppressor from each particular virus. Multiplecopies of a suppressor may be used. Different suppressors may be usedtogether (e.g., in tandem).

RNA Molecules

Essentially any RNA molecule which is desirable to be expressed in aplant seed can be co-expressed with the silencing suppressor. Theencoded polypeptides may be involved in metabolism of oil, starch,carbohydrates, nutrients, etc., or may be responsible for the synthesisof proteins, peptides, fatty acids, lipids, waxes, oils, starches,sugars, carbohydrates, flavors, odors, toxins, carotenoids. hormones,polymers, flavonoids, storage proteins, phenolic acids, alkaloids,lignins, tannins, celluloses, glycoproteins, glycolipids, etc,preferably the biosynthesis or assembly of TAG.

In a particular example, the plants produced increased levels of enzymesfor oil production in plants such as Brassicas, for example canola orsunflower, safflower, flax, cotton, soya bean, Camelina or maize.

Levels of LC-PUFA Produced

The levels of the LC-PUFA or combination of LC-PUFAs that are producedin the recombinant cell or plant part such as seed are of importance.The levels may be expressed as a composition (in percent) of the totalfatty acid that is a particular LC-PUFA or group of related LC-PUFA, forexample the ω3 LC-PUFA or the ω6 LC-PUFA, or the VLC-PUFA, or otherwhich may be determined by methods known in the art. The level may alsobe expressed as a LC-PUFA content, such as for example the percentage ofLC-PUFA in the dry weight of material comprising the recombinant cells,for example the percentage of the weight of seed that is LC-PUFA. Itwill be appreciated that the LC-PUFA that is produced in an oilseed maybe considerably higher in terms of LC-PUFA content than in a vegetableor a grain that is not grown for oil production, yet both may havesimilar LC-PUFA compositions, and both may be used as sources of LC-PUFAfor human or animal consumption.

The levels of LC-PUFA may be determined by any of the methods known inthe art. In a preferred method, total lipid is extracted from the cells,tissues or organisms and the fatty acid converted to methyl estersbefore analysis by gas chromatography (GC). Such techniques aredescribed in Example 1. The peak position in the chromatogram may beused to identify each particular fatty acid, and the area under eachpeak integrated to determine the amount. As used herein, unless statedto the contrary, the percentage of particular fatty acid in a sample isdetermined as the area under the peak for that fatty acid as apercentage of the total area for fatty acids in the chromatogram. Thiscorresponds essentially to a weight percentage (w/w). The identity offatty acids may be confirmed by GC-MS. Total lipid may be separated bytechniques known in the art to purify fractions such as the TAGfraction. For example, thin-layer chromatography (TLC) may be performedat an analytical scale to separate TAG from other lipid fractions suchas DAG, acyl-CoAs or phospholipid in order to determine the fatty acidcomposition specifically of TAG.

In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fattyacids in the extracted lipid is between about 21% and about 40% of thetotal fatty acids in the cell. In a further embodiment, the total fattyacid in the cell has less than 1% C20:1. In preferred embodiments, theextractable TAG in the cell comprises the fatty acids at the levelsreferred to herein. Each possible combination of the features definingthe lipid as described herein is also encompassed.

The level of production of LC-PUFA in the recombinant cell, plant orplant part such as seed may also be expressed as a conversion percentageof a specific substrate fatty acid to one or more product fatty acids,which is also referred to herein as a “conversion efficiency” or“enzymatic efficiency”. This parameter is based on the fatty acidcomposition in the lipid extracted from the cell, plant, plant part orseed, i.e., the amount of the LC-PUFA formed (including other LC-PUFAderived therefrom) as a percentage of one or more substrate fatty acids(including all other fatty acids derived therefrom). The general formulafor a conversion percentage is: 100×(the sum of percentages of theproduct LC-PUFA and all products derived therefrom)/(the sum of thepercentages of the substrate fatty acid and all products derivedtherefrom). With regard to DHA, for example, this may be expressed asthe ratio of the level of DHA (as a percentage in the total fatty acidcontent in the lipid) to the level of a substrate fatty acid (e.g. OA,LA, ALA, SDA, ETA or EPA) and all products including DHA derived fromthe substrate. The conversion percentage or efficiency of conversion canbe expressed for a single enzymatic step in a pathway, or for part orthe whole of a pathway.

Specific conversion efficiencies are calculated herein according to theformulae:

-   1. OA to DHA=100×(% DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA,    ALA, SDA, ETrA, ETA, EPA, DPA and DHA).-   2. LA to DHA=100×(% DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA,    SDA, ETrA, ETA, EPA, DPA and DHA).-   3. ALA to DHA=100×(% DHA)/(sum % for ALA, SDA, ETrA, ETA, EPA, DPA    and DHA).-   4. EPA to DHA=100×(% DHA)/(sum % for EPA, DPA and DHA).-   5. DPA to DHA (Δ4-desaturase efficiency)=100×(% DHA)/(sum % for DPA    and DHA).-   6. Δ12-desaturase efficiency=100×(sum % for LA, GLA, DGLA, ARA, EDA,    ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for OA, LA, GLA, DGLA,    ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).-   7. ω3-desaturase efficiency=100×(sum % for ALA, SDA, ETrA, ETA, EPA,    DPA and DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA,    ETA, EPA, DPA and DHA).-   8. OA to ALA=100×(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and    DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA,    EPA, DPA and DHA).-   9. Δ6-desaturase efficiency (on ω3 substrate ALA)=100×(sum % for    SDA, ETA, EPA, DPA and DHA)/(% ALA, SDA, ETrA, ETA, EPA, DPA and    DHA).-   10. Δ6-elongase efficiency (on ω3 substrate SDA)=100×(sum % for ETA,    EPA, DPA and DHA)/(sum % for SDA, ETA, EPA, DPA and DHA).-   11. Δ5-desaturase efficiency (on ω3 substrate ETA)=100×(sum % for    EPA, DPA and DHA)/(sum % for ETA, EPA, DPA and DHA).-   12. Δ5-elongase efficiency (on ω3 substrate EPA)=100×(sum % for DPA    and DHA)/(sum % for EPA, DPA and DHA).

The fatty acid composition of the lipid, preferably seedoil, of theinvention, is also characterised by the ratio of ω6 fatty acids:ω3 fattyacids in the total fatty acid content, for either total ω6 fattyacids:total ω3 fatty acids or for new ω6 fatty acids:new ω3 fatty acids.The terms total ω6 fatty acids, total ω3 fatty acids, new ω6 fatty acidsand new ω3 fatty acids have the meanings as defined herein. The ratiosare calculated from the fatty acid composition in the lipid extractedfrom the cell, plant, plant part or seed, in the manner as exemplifiedherein. It is desirable to have a greater level of ω3 than ω6 fattyacids in the lipid, and therefore an ω6:ωratio of less than 1.0 ispreferred. A ratio of 0.0 indicates a complete absence of the defined ω6fatty acids; a ratio of 0.03 was achieved. Such low ratios can beachieved through the combined use of a Δ6-desaturase which has an ω3substrate preference together with an ω3-desaturase, particularly afungal ω3-desaturase such as the Pichia pastoris ω3-desaturase asexemplified herein.

The yield of LC-PUFA per weight of seed may also be calculated based onthe total oil content in the seed and the % DHA and/or DPA in the oil.For example, if the oil content of canola seed is about 40% (w/w) andabout 12% of the total fatty acid content of the oil is DHA, the DHAcontent of the seed is about 4.8% or about 48 mg per gram of seed. Asdescribed in Example 2, the DHA content of Arabidopsis seed having about9% DHA, which has a lower oil content than canola, was about 25 mg/gseed. At a DHA content of about 21%, canola seed or Camelina sativa seedhas a DHA content of about 84 mg per gram of seed. The present inventiontherefore provides Brassica napus, B. juncea and Camelina sativa plants,and seed obtained therefrom, comprising at least about 80 mg or at leastabout 84 mg DHA per gram seed. The seed has a moisture content as isstandard for harvested mature seed after drying down (4-15% moisture).The invention also provides a process for obtaining oil, comprisingobtaining the seed and extracting the oil from the seed, and uses of theoil and methods of obtaining the seed comprising harvesting the seedsfrom the plants according to the invention.

The amount of DHA and/or DPA produced per hectare can also be calculatedif the seed yield per hectare is known or can be estimated. For example,canola in Australia typically yields about 2.5 tonnes seed per hectare,which at 40% oil content yields about 1000 kg of oil. At 20.1% DHAand/or DPA in the total oil, this provides about 200 kg of DHA and/orDPA per hectare. If the oil content is reduced by 50%, this stillprovides about 100 kg DHA and/or DPA/ha.

Evidence to date suggests that some desaturases expressed heterologouslyin yeast or plants have relatively low activity in combination with someelongases. This may be alleviated by providing a desaturase with thecapacity of to use an acyl-CoA form of the fatty acid as a substrate inLC-PUFA synthesis, and this is thought to be advantageous in recombinantcells particularly in plant cells. A particularly advantageouscombination for efficient DHA and/or DPA synthesis is a fungalω3-desaturase, for example such as the Pichia pastoris ω3-desaturase(SEQ ID NO: 6), with a Δ6-desaturase which has a preference for ω3 acylsubstrates such as, for example, the Micromonas pusilla Δ6-desaturase(SEQ ID NO: 9), or variants thereof which have at least 95% amino acidsequence identity.

As used herein, the term “essentially free” means that the composition(for example lipid or oil) comprises little (for example, less thanabout 0.5%, less than about 0.25%, less than about 0.1%, or less thanabout 0.01%) or none of the defined component. In an embodiment,“essentially free” means that the component is undetectable using aroutine analytical technique, for example a specific fatty acid (such asω6-docosapentaenoic acid) cannot be detected using gas chromatography asoutlined in Example 1.

Production of Oils

Techniques that are routinely practiced in the art can be used toextract, process, and analyze the oils produced by cells, plants, seeds,etc of the instant invention. Typically, plant seeds are cooked,pressed, and extracted to produce crude oil, which is then degummed,refined, bleached, and deodorized. Generally, techniques for crushingseed are known in the art. For example, oilseeds can be tempered byspraying them with water to raise the moisture content to, e.g., 8.5%,and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm.Depending on the type of seed, water may not be added prior to crushing.Application of heat deactivates enzymes, facilitates further cellrupturing, coalesces the oil droplets, and agglomerates proteinparticles, all of which facilitate the extraction process.

In an embodiment, the majority of the seed oil is released by passagethrough a screw press. Cakes expelled from the screw press are thensolvent extracted, e.g., with hexane, using a heat traced column.Alternatively, crude oil produced by the pressing operation can bepassed through a settling tank with a slotted wire drainage top toremove the solids that are expressed with the oil during the pressingoperation. The clarified oil can be passed through a plate and framefilter to remove any remaining fine solid particles. If desired, the oilrecovered from the extraction process can be combined with the clarifiedoil to produce a blended crude oil.

Once the solvent is stripped from the crude oil, the pressed andextracted portions are combined and subjected to normal oil processingprocedures. As used herein, the term “purified” when used in connectionwith lipid or oil of the invention typically means that that theextracted lipid or oil has been subjected to one or more processingsteps of increase the purity of the lipid/oil component. For example, apurification step may comprise one or more or all of the groupconsisting of: degumming, deodorising, decolourising, drying and/orfractionating the extracted oil. However, as used herein, the term“purified” does not include a transesterification process or otherprocess which alters the fatty acid composition of the lipid or oil ofthe invention so as to increase the DHA content as a percentage of thetotal fatty acid content. Expressed in other words, the fatty acidcomposition of the purified lipid or oil is essentially the same as thatof the unpurified lipid or oil.

Degumming

Degumming is an early step in the refining of oils and its primarypurpose is the removal of most of the phospholipids from the oil, whichmay be present as approximately 1-2% of the total extracted lipid.Addition of ˜2% of water, typically containing phosphoric acid, at70-80° C. to the crude oil results in the separation of most of thephospholipids accompanied by trace metals and pigments. The insolublematerial that is removed is mainly a mixture of phospholipids andtriacylglycerols and is also known as lecithin. Degumming can beperformed by addition of concentrated phosphoric acid to the crudeseedoil to convert non-hydratable phosphatides to a hydratable form, andto chelate minor metals that are present. Gum is separated from theseedoil by centrifugation.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil,sometimes also referred to as neutralization. It usually followsdegumming and precedes bleaching. Following degumming, the seedoil cantreated by the addition of a sufficient amount of an alkali solution totitrate all of the fatty acids and phosphoric acids, and removing thesoaps thus formed. Suitable alkaline materials include sodium hydroxide,potassium hydroxide, sodium carbonate, lithium hydroxide, calciumhydroxide, calcium carbonate and ammonium hydroxide. This process istypically carried out at room temperature and removes the free fattyacid fraction. Soap is removed by centrifugation or by extraction into asolvent for the soap, and the neutralised oil is washed with water. Ifrequired, any excess alkali in the oil may be neutralized with asuitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C.for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and inthe absence of oxygen by operating with nitrogen or steam or in avacuum. This step in oil processing is designed to remove unwantedpigments (carotenoids, chlorophyll, gossypol etc), and the process alsoremoves oxidation products, trace metals, sulphur compounds and tracesof soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature(200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achievedby introducing steam into the seedoil at a rate of about 0.1ml/minute/100 nil of seedoil. After about 30 minutes of sparging, theseedoil is allowed to cool under vacuum. The seedoil is typicallytransferred to a glass container and flushed with argon before beingstored under refrigeration. This treatment improves the colour of theseedoil and removes a majority of the volatile substances or odorouscompounds including any remaining free fatty acids, monoacylglycerolsand oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production ofoils for the separation of oils and fats into solid (stearin) and liquid(olein) fractions by crystallization at sub-ambient temperatures. It wasapplied originally to cottonseed oil to produce a solid-free product. Itis typically used to decrease the saturated fatty acid content of oils.

Transesterification

As used herein, “transesterification” means a process that exchanges thefatty acids within and between TAGs or transfers the fatty acids toanother alcohol to form an ester. This may initially involve releasingfatty acids from the TAGs as free fatty acids or it may directly producefatty acid esters, preferably fatty acid methyl esters or ethyl esters.In a transesterification reaction of the TAG with an alcohol such asmethanol or ethanol, the alkyl group of the alcohol forms an esterlinkage with the acyl groups (including the DHA) of the TAG. Whencombined with a fractionation process, transesterification can be usedto modify the fatty acid composition of lipids (Marangoni et al., 1995).Transesterification can use either chemical (e.g. strong acid or basecatalysed) or enzymatic means, the latter using lipases which may beposition-specific (sn-1/3 or sn-2 specific) for the fatty acid on theTAG, or having a preference for some fatty acids over others (Speranzaet al, 2012). The fatty acid fractionation to increase the concentrationof LC-PUFA in an oil can be achieved by any of the methods known in theart, such as, for example, freezing crystallization, complex formationusing urea, molecular distillation, supercritical fluid extraction,counter current chromatography and silver ion complexing. Complexformation with urea is a preferred method for its simplicity andefficiency in reducing the level of saturated and monounsaturated fattyacids in the oil (Gamez et al., 2003). Initially, the TAGs of the oilare split into their constituent fatty acids, often in the form of fattyacid esters, by hydrolysis under either acid or base catalysed reactionconditions, whereby one mol of TAG is reacted with at least 3 mol ofalcohol (e.g. ethanol for ethyl esters or methanol for methyl esters)with excess alcohol used to enable separation of the formed alkyl estersand the glycerol that is also formed, or by lipases. These free fattyacids or fatty acid esters, which are usually unaltered in fatty acidcomposition by the treatment, may then be mixed with an ethanolicsolution of urea for complex formation. The saturated andmonounsaturated fatty acids easily complex with urea and crystallize outon cooling and may subsequently be removed by filtration. The non-ureacomplexed fraction is thereby enriched with LC-PUFA.

Feedstuffs

The present invention includes compositions which can be used asfeedstuffs. For purposes of the present invention, “feedstuffs” includeany food or preparation for human or animal consumption which when takeninto the body (a) serve to nourish or build up tissues or supply energy;and/or (b) maintain, restore or support adequate nutritional status ormetabolic function. Feedstuffs of the invention include nutritionalcompositions for babies and/or young children such as, for example,infant formula, and seedmeal of the invention.

Feedstuffs of the invention comprise, for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product ofthe method of the invention, the product of the fermentation process ofthe invention, or a composition along with a suitable carrier(s). Theterm “carrier” is used in its broadest sense to encompass any componentwhich may or may not have nutritional value. As the skilled addresseewill appreciate, the carrier must be suitable for use (or used in asufficiently low concentration) in a feedstuff such that it does nothave deleterious effect on an organism which consumes the feedstuff.

The feedstuff of the present invention comprises an oil, fatty acidester, or fatty acid produced directly or indirectly by use of themethods, cells or plants disclosed herein. The composition may either bein a solid or liquid form. Additionally, the composition may includeedible macronutrients, protein, carbohydrate, vitamins, and/or mineralsin amounts desired for a particular use. The amounts of theseingredients will vary depending on whether the composition is intendedfor use with normal individuals or for use with individuals havingspecialized needs, such as individuals suffering from metabolicdisorders and the like.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and diglycerides. Examples of such carbohydrates include (but arenot limited to): glucose, edible lactose, and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include (but are not limitedto) soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention: calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material or by de novo synthesis.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including (but notlimited to): margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

Additionally, fatty acids produced in accordance with the presentinvention or host cells transformed to contain and express the subjectgenes may also be used as animal food supplements to alter an animal'stissue, egg or milk fatty acid composition to one more desirable forhuman or animal consumption. Examples of such animals include sheep,cattle, horses, poultry such as chickens and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish or crustaceans such as, forexample, prawns for human or animal consumption. Preferred fish aresalmon.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves and stems which may be used directly as foodor feed for humans or other animals. For example, animals may grazedirectly on such plants grown in the field or be fed more measuredamounts in controlled feeding. The invention includes the use of suchplants and plant parts as feed for increasing the LC-PUFA levels inhumans and other animals.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more of the fatty acidsand/or resulting oils produced using the methods of the invention,preferably in the form of ethyl esters of the fatty acids.

A pharmaceutical composition may comprise one or more of the fatty acidsand/or oils, in combination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquidor powder, injectible, or topical ointment or cream. Proper fluidity canbe maintained, for example, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. It mayalso be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, sweetening agents, flavoring agentsand perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanthor mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, fatty acids produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant fatty acid(s).

For intravenous administration, the fatty acids produced in accordancewith the present invention or derivatives thereof may be incorporatedinto commercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid, especially LC-PUFA, is desirable.However, it will be appreciated that any amount of fatty acid will bebeneficial to the subject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include, for example, enteral (e.g., oral andrectal) and parenteral. For example, a liquid preparation may beadministered orally or rectally. Additionally, a homogenous mixture canbe completely dispersed in water, admixed under sterile conditions withphysiologically acceptable diluents, preservatives, buffers orpropellants to form a spray or inhalant.

The dosage of the composition to be administered to the patient may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight of the patient, age of the patient, overallhealth of the patient, past history of the patient, immune status of thepatient, etc.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. It may be added to pre-existing cosmeticcompositions such that a mixture is formed or a fatty acid producedaccording to the subject invention may be used as the sole “active”ingredient in a cosmetic composition.

EXAMPLES Example 1. Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

Exogenous genetic constructs were expressed in plant cells in atransient expression system essentially as described by Voinnet et al.(2003) and Wood et al. (2009).

Gas Chromatography (GC) Analysis of Fatty Acids

FAME were analysed by gas chromatography using an Agilent Technologies7890A GC (Palo Alto, Calif., USA) equipped with a 30 m SGE-BPX70 column(70% cyanopropyl polysilphenylene-siloxane, 0.25 mm inner diameter, 0.25mm film thickness), an FID, a split/splitless injector and an AgilentTechnologies 7693 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in split mode (50:1 ratio) at anoven temperature of 150° C. After injection, the oven temperature washeld at 150° C. for 1 min then raised to 210° C. at 3° C. min⁻¹, againraised to 240° C. at 50° C. min′ and finally holding for 1.4 mM at 240°C. Peaks were quantified with Agilent Technologies ChemStation software(Rev B.Δ4.03 (16), Palo Alto, Calif., USA) based on the response of theknown amount of the external standard GLC-411 (Nucheck) and C17:0-MEinternal standard.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Lipids

Total lipids were extracted from freeze-dried developing seeds, twelvedays after flowering (daf), and mature seeds after adding a known amountof tri-C17:0-TAG as an internal quantitation standard. The extractedlipids were dissolved into 1 mL of 10 mM butylated hydroxytoluene inbutanol:methanol (1:1 v/v) per 5 mg dry material and analysed using anAgilent 1200 series LC and 6410b electrospray ionisation triplequadrupole LC-MS. Lipids were chromatographically separated using anAscentis Express RP-Amide column (50 mm×2.1 mm, 2.7 μm, Supelco)operating a binary gradient with a flow rate of 0.2 mL/min. The mobilephases were: A. 10 mM ammonium formate in H₂O:methanol: tetrahydrofuran(50:20:30 v/v/v); B. 10 mM ammonium formate in H₂O:methanol:tetrahydrofuran (5:20:75, v/v/v). Multiple reaction monitoring (MRM)lists were based on the following major fatty acids: 16:0, 18:0, 18:1,18:2, 18:3, 18:4, 20:1, 20:2, 20:3, 20:4, 20:5, 22:4, 22:5, 22:6 using acollision energy of 30 V and fragmentor of 60 V. Individual MRM TAG wasidentified based on ammoniated precursor ion and product ion fromneutral loss of 22:6. TAG was quantified using a 10 μM tristearinexternal standard.

Lipid Profiling with LC-MS

The extracted total lipids were analysed using an Agilent 1200 series LCcoupled to an Agilent 6410B electrospray ionisation QQQ-MS (Agilent,Palo Alto, Calif., USA). A 5 μL injection of each total lipid extractwas chromatographically separated with an Ascentis Express RP-Amide 50mm×2.1 mm, 2.7 μm HPLC column (Sigma-Aldrich, Castle Hill, Australia)using a binary gradient with a flow rate of 0.2 ml/min. The mobilephases were: A. 10 mM ammonium formate in H₂O:methanol:tetrahydrofuran(50:20:30, v/v/v.); B. 10 mM ammonium formate inH₂O:methanol:tetrahydrofuran (5:20:75, v/v/v.). Selected neutral lipids(TAG and DAG) and phospholipids (PL, including PC, PE, PI, PS, PA, PG)were analysed by multiple reaction monitoring (MRM) using a collisionenergy of 30 V and fragmentation energy of 60 V. Neutral lipids weretargeted on the following major fatty acids: 16:0 (palmitic acid), 18:0(stearic acid), 18:1ω9 (oleic acid, OA), 18:2ω6 (linoleic acid, LA),18:3ω3 (α-linolenic acid, ALA), 18:4ω3 (stearidonic acid, SDA), 20:1,20:2, 20:3, 20:4ω3, 20:5ω3, 22:4ω3, 22:5ω3, 22:6ω3, while phospholipidswere scanned containing C₁₆, C₁₈, C₂₀ and C₂₂ species with double bondsof 0-3, 0-4, 0-5, 4-6 respectively.

Individual MRM TAG was identified based on ammoniated precursor ion andproduct ion from neutral loss of 20:1, SDA, EPA and DHA. TAG and DAGwere quantified using the 50 μM tristearin and distearin as externalstandards. PL were quantified with 10 uM of di-18:0-PC, di-17:0-PA,di-17:0-PE, 17:0-17:1-PG, di-18:1-PI and di-17:0-PS external standards(Avanti Polar Lipids, Alabaster, Ala., USA). Selected TAG, DAG and PLspecies were further confirmed by Agilent 6520 Q-TOF MS/MS.

Determination of Seed Fatty Acid Profile and Oil Content

Where seed oil content was to be determined, seeds were dried in adesiccator for 24 h and approximately 4 mg of seed was transferred to a2 ml glass vial containing Teflon-lined screw cap. 0.05 mgtriheptadecanoin dissolved in 0.1 ml toluene was added to the vial asinternal standard.

Seed FAME were prepared by adding 0.7 ml of 1N methanolic HCl (Supelco)to the vial containing seed material, vortexed briefly and incubated at80° C. for 2 h. After cooling to room temperature, 0.3 ml of 0.9% NaCl(w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 minin Heidolph Vibramax 110. The FAME was collected into 0.3 ml glassinsert and analysed by GC with a flame ionization detector (FID) asmentioned earlier.

The peak area of individual FAME were first corrected on the basis ofthe peak area responses of known amount of the same FAMEs present in acommercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 containsequal amounts of 31 fatty acids (% by wt), ranging from C8:0 to C22:6.In case of fatty acids, which were not present in the standard, theinventors took the peak area responses of the most similar FAME. Forexample, peak area response of FAMEs of 16:1d9 was used for 16:1d7 andFAME response of C22:6 was used for C22:5. The corrected areas were usedto calculate the mass of each FAME in the sample by comparison to theinternal standard mass. Oil is stored mainly in the form of TAG and itsweight was calculated based on FAME weight. Total moles of glycerol wasdetermined by calculating moles of each FAMES and dividing total molesof FAMES by three. TAG was calculated as the sum of glycerol and fattyacyl moieties using a relation: % oil by weight=100×((41×total molFAME/3)+(total g FAME−(15×total mol FAME)))/g seed, where 41 and 15 aremolecular weights of glycerol moiety and methyl group, respectively.

Analysis of the Sterol Content of Oil Samples

Samples of approximately 10 mg of oil together with an added aliquot ofC24:0 monol as an internal standard were saponified using 4 mL 5% KOH in80% MeOH and heating for 2 h at 80° C. in a Teflon-lined screw-cappedglass tube. After the reaction mixture was cooled, 2 mL of Milli-Q waterwere added and the sterols were extracted into 2 mL of hexane:dichloromethane (4:1 v/v) by shaking and vortexing. The mixture wascentrifuged and the sterol extract was removed and washed with 2 mL ofMilli-Q water. The sterol extract was then removed after shaking andcentrifugation. The extract was evaporated using a stream of nitrogengas and the sterols silylated using 200 mL of BSTFA and heating for 2 hat 80° C.

For GC/GC-MS analysis of the sterols, sterol-OTMSi derivatives weredried under a stream of nitrogen gas on a heat block at 40° C. and thenre-dissolved in chloroform or hexane immediately prior to GC/GC-MSanalysis. The sterol-OTMS derivatives were analysed by gaschromatography (GC) using an Agilent Technologies 6890A GC (Palo Alto,Calif., USA) fitted with an Supelco Equity™-1 fused silica capillarycolumn (15 m×0.1 mm i.d., 0.1 μm film thickness), an FID, asplit/splitless injector and an Agilent Technologies 7683B Series autosampler and injector. Helium was the carrier gas. Samples were injectedin splitless mode at an oven temperature of 120° C. After injection, theoven temperature was raised to 270° C. at 10° C. min⁻¹ and finally to300° C. at 5° C. min⁻¹. Peaks were quantified with Agilent TechnologiesChemStation software (Palo Alto, Calif., USA). GC results are subject toan error of ±5% of individual component areas.

GC-mass spectrometric (GC-MS) analyses were performed on a FinniganThermoquest GCQ GC-MS and a Finnigan Thermo Electron Corporation GC-MS;both systems were fitted with an on-column injector and ThermoquestXcalibur software (Austin, Tex., USA). Each GC was fitted with acapillary column of similar polarity to that described above. Individualcomponents were identified using mass spectral data and by comparingretention time data with those obtained for authentic and laboratorystandards. A full procedural blank analysis was performed concurrent tothe sample batch.

RT-PCR Conditions

Reverse transcription-PCR (RT-PCR) amplification was typically carriedout using the Superscript III One-Step RT-PCR system (Invitrogen) in avolume of 25 using 10 pmol of the forward primer and 30 pmol of thereverse primer, MgSO₄ to a final concentration of 2.5 mM, 400 ng oftotal RNA with buffer and nucleotide components according to themanufacturer's instructions. Typical temperature regimes were: 1 cycleof 45° C. for 30 minutes for the reverse transcription to occur; then 1cycle of 94° C. for 2 minutes followed by 40 cycles of 94° C. for 30seconds, 52° C. for 30 seconds, 70° C. for 1 minute; then 1 cycle of 72°C. for 2 minutes before cooling the reaction mixtures to 5° C.

Determination of Copy-Number of Transgenes by Digital PCR

To determine the copy-number of transgenes in a transgenic plant, adigital PCR method was used as follows. This method could also be usedto determine whether a plant was transgenic for the genetic constructsdescribed herein. About a centimetre square of leaf tissue was harvestedfrom each individual plant and placed in a collection microtube(Qiagen). The samples were then freeze dried for 24 to 48 hr. Forbreaking up the samples for DNA extraction, stainless steel ballbearings were added to each dried sample and the tubes shaken on aQiagen Tissue lyser. 3754 of extraction buffer (0.1M Tris-HCl pH8, 0.05MEDTA pH8 and 1.25% SDS) was added to each tube, the mixtures incubatedat 65° C. for 1 hr, and then cooled before 187 μL of 6M ammonium acetate(4° C.) was added to each tube with thorough mixing. The samples werethen centrifuged for 30 min at 3000 rpm. The supernatant from each tubewas removed into new microtubes each containing 220 μL of isopropanolfor precipitation of the DNA at room temperature for 5 min. DNA wascollected by centrifuging the tubes at 3000 rpm for 30 min, the DNApellets washed with 320 μL of 70% ethanol and dried before resuspensionof the DNA in 225 μL of water. Non-dissolved material was pelleted bycentrifugation at 3000 rpm for 20 min, and 1504, of each supernatanttransferred to 96-well plates for long term storage.

For efficient and quantitative digital PCR (ddPCR) the DNA was digestedwith restriction enzymes prior to amplification reactions, to ensurethat multiple copies of the transgenes or multiple insertions werephysically separated. Aliquots of the DNA preparations were thereforedigested with EcoRI and BamHI, together, in 204, volumes using 10×EcoRIbuffer, 5 μL of DNA and about 4 units of each enzyme per sample,incubated overnight at 37° C.

The primers used in these PCR reactions were designed using Primer3software to confirm that primers for the reference and target genes werenot predicted to interact, or such interaction would not be a problemunder the conditions used. The reference gene used in the assay was thecanola Hmg (high mobility group) gene, present at one gene per canolagenome (Weng et al., 2004). Since canola is an allotetraploid, it wastaken that there were 4 copies of the Hmg gene, i.e. 2 alleles of eachof the two genes, in Brassica napus. The reference gene reactions usedthe pair of primers and a dual-labelled probe, as follows: Sense primer,Can11 GCGAAGCACATCGAGTCA (SEQ ID NO:50); Antisense primer, Can12GGTTGAGGTGGTAGCTGAGG (SEQ ID NO:51); Probe, Hmg-P35′-Hex/TCTCTAC/zen/CCGTCTCACATGACGC/3IABkFQ/-3′ (SEQ ID NO:52). Theamplification product size was 73 bp.

In one target gene amplification reaction which detected a region of thePPT selectable marker gene to screen all of the transgenic plants, thesense primer was Can17, ATACAAGCACGGTGGATGG (SEQ ID NO:53); theantisense primer, Can18 TGGTCTAACAGGTCTAGGAGGA (SEQ ID NO:54); theprobe, PPT-P3 5′-/FAM/TGGCAAAGA/zen/GATTTCGAGCTTCCTGC/3IABkFQ/-3′ (SEQID NO:55). The size of this target gene amplification product was 82 bp.On some occasions, a second target gene assay was performed in parallelto detect partial insertions of the T-DNA. This second assay detected aregion of the Δ6-desaturase gene using a sense primer, Can23CAAGCACCGTAGTAAGAGAGCA (SEQ ID NO:56), the antisense primer, Can24CAGACAGCCTGAGGTTAGCA (SEQ ID NO:57); the probe, Diodes-P35′-/FAM/TCCCCACTT/zen/CTTAGCGAAAGGAACGA/3IABkFQ/-3′ (SEQ ID NO:58). Thesize of this target gene amplification product was 89 bp. Reactionsroutinely used 2 μL of the digested DNA preparations. Reactioncomposition per sample: reference sense primer (10 pM), 1 μL; referenceantisense primer (10 pM), 1 μL; reference gene probe (10 pM), 0.5 μL;target gene sense primer (10 pM), 1 μL; target gene antisense primer (10pM), 1 μL; target gene probe (10 pM), 0.544 ddPCR reagent mix, 12.5 μL;water 5.5 μL in a total volume of 25 μL.

The mixtures were then placed into a QX100 droplet generator, whichpartitioned each sample into 20000 nanoliter-sized droplets. This wasdone in 8-well cartridges until all of the samples were processed andtransferred to a 96-well PCR plate. This plate was then heat sealed witha pierceable foil using a plate sealer machine. The samples were thentreated under the following reaction conditions: 95° C., 10 min, rampingat 2.5° C./s; then 39 cycles of 94° C., 30 s ramping at 2.5° C./s; 61°C., 1 min, ramping at 2.5° C./s; 98° C., 10 min, followed by cooling to12° C. Following the amplification reactions of the DNA in the droplets,the plate was placed in a QX100 droplet reader which analysed eachdroplet individually using a two-color detection system (set to detectFAM or Hex). The droplet digital PCR data were viewed as either a 1-Dplot with each droplet from a sample plotted on the graph offluorescence intensity, or a 2-D plot in which fluorescence (FAM) wasplotted against fluorescence (Hex) for each droplet. The softwaremeasured the number of positive and negatives droplets for eachfluorophore (FAM or Hex) in each sample. The software then fitted thefraction of positive droplets to a Poisson algorithm to determine theconcentration of the target DNA molecule in units of copies/μL input.The copy number variation was calculated using the formula:CNV=(AJB)*Nb, where A=concentration of target gene, B=concentration ofreference gene, and Nb=4, the number of copies of the reference gene inthe genome.

Assessment of Pollen Viability

Fluorescein diacetate (FDA) was dissolved in acetone at 2 mg/ml toprovide a stock solution. FDA dilutions were prepared just before use byadding drops of the FDA stock solution to 2 ml of a sucrose solution(0.5 M) until saturation was reached as indicated by the appearance ofpersistent cloudiness.

Propidium iodide (PI) was dissolved in sterile distilled water at 1mg/ml to provide a stock solution. Just before use, 100 μL of the stocksolution was added to 10 ml of sterile distilled water to make a workingsolution. To check the ratio of viable and non-viable pollen, PI and FDAstock solutions were mixed in 2:3 ratio.

Transgenic and wild-type canola and mustard plants were grown understandard conditions in a glasshouse at 22±2° C. with a 16 hr photoperiodper day. Mature flower buds which were ready to open in the next daywere labelled and collected on the following morning at 9-10 am. Pollenfrom opened flowers were stained with the FDA/PI mixture and visualizedusing a Leica MZFLIII fluorescence microscope. GFP-2, a 510 nm long passemission filter (transmitting red and green light) with a 480/40 nmexcitation filter was used to detect viable and non-viable pollen.Non-viable pollen which took up the PI stain appeared red under thefluorescence microscope whereas viable pollen appeared bright green whenstained with PI and FDA.

Example 2. Stable Expression of Transgenic DHA Pathways in Arabidopsisthaliana Seeds

Binary Vector Construction

The binary vectors pJP3416-GA7 (also referred to herein as “GA7”described in WO 2013/185184) and pJP3404 each contained sevenheterologous fatty acid biosynthesis genes, encoding 5 desaturases and 2elongases, and a plant selectable marker between the left and rightborder repeats of the T-DNA present in each vector (FIGS. 2 and 3). SEQID NO:1 provides the nucleotide sequence of the T-DNA region ofpJP3416-GA7 from the right to left border sequences. Both geneticconstructs contained plant codon-optimised genes encoding a Lachanceakluyveri Δ12-desaturase (comprising nucleotides 14143-16648 of SEQ IDNO:1), a Pichia pastoris ω3-desaturase (comprising nucleotides7654-10156 of SEQ ID NO:1), a Micromonas pusilla Δ6-desaturase(comprising nucleotides 226-2309 of SEQ ID NO:1), Pavlova salina Δ5- andΔ4-desaturases (comprising nucleotides 4524-6485 and 10157-14142 of SEQID NO:1, respectively) and Pyramimonas cordata Δ6- and Δ5-elongases(comprising nucleotides 2310-4523 and 17825-19967 of SEQ ID NO:1,respectively).

The seven coding regions in the constructs were each under the controlof a seed specific promoter-three different promoters were used, namelythe truncated Brassica napus napin promoter (pBnFP1), the Arabidopsisthaliana FAE1 promoter (pAtFAE1) and the Linum usitatissimum conlinin 1promoter (pLuCnl1). The seven fatty acid biosynthesis genes togethercoded for an entire DHA synthesis pathway that was designed to convert18:1^(Δ9) (oleic acid) through to 22:6^(Δ4,7,10,13,16,19) (DHA). Bothbinary vectors contained a BAR plant selectable marker coding regionoperably linked to a Cauliflower Mosaic Virus (CaMV) 35S promoter withduplicated enhancer region and A. tumefaciens nos3′ polyadenylationregion-transcription terminator. The plant selectable marker wassituated adjacent to the left border of the T-DNA region, thereforedistally located on the T-DNA with respect to the orientation of T-DNAtransfer into the plant cells. This increased the likelihood thatpartial transfer of the T-DNA, which would be likely to not include theselectable marker gene, would not be selected. pJP3416-GA7 and pJP3404each contained an RiA4 origin of replication from Agrobacteriumrhizogenes (Hamilton, 1997).

The GA7 construct also included two Nicotiana tabacum Rb7 matrixattachment region (MAR) sequences, as described by Hall et al. (1991).MAR sequences, sometimes termed nuclear attachment regions, are known tobind specifically to the nuclear matrix in vitro and may mediate bindingof chromatin to the nuclear matrix in vivo. MARs are thought to functionto reduce transgene silencing. In pJP3416-GA7 the MARs were alsoinserted and positioned within the T-DNA region in order to act as DNAspacers to insulate transgenic expression cassettes. The pJP3416 vectorprior to insertion of the GA7 region contained only the plant selectablemarker cassette between the borders.

A. thaliana Transformation and Analysis of Fatty Acid Composition

The chimeric vectors were introduced into A. tumefaciens strain AGL1 andcells from cultures of the transformed Agrobacterium used to treat A.thaliana (ecotypes Columbia and a fad2 mutant) plants using the floraldip method for transformation (Clough and Bent, 1998). After maturation,the T₁ seeds from the treated plants were harvested and plated onto MSplates containing PPT for selection of plants containing the BARselectable marker gene. Surviving, healthy T₁ seedlings were transferredto soil. After growth of the plants to maturity and allowing forself-fertilisation, T₂ seeds from these plants were harvested and thefatty acid composition of their seed lipid analysed by GC analysis asdescribed in Example 1.

The pJP3416-GA7 construct resulted in the production of slightly higherlevels of DHA, as a percentage of total fatty acid content, on averagethan the pJP3404 construct. The conversion efficiencies for eachenzymatic step in the production of DHA from oleic acid were calculatedas (% products×100)/(% remaining substrate+% products), therebyexpressed as a percentage.

The highest observed level of DHA produced in the pJP3416-GA7 T2transformed lines was 6.2%, additionally with 0.5% EPA and 0.2% DPA(line #14). These T2 seeds were still segregating for the transgene i.e.were not yet uniformly homozygous. The level of ω3 fatty acids producedas a result of the transgenes in these seeds (total new ω3 fatty acids,excluding the level of ALA which was produced endogenously in theColumbia background) was 10.7% while the level of ω6 fatty acids (totalnew ω6 fatty acids but excluding 18:2^(Δ9,12)) was 1.5%. This representsan extremely favourable ration of new ω3 fatty acids:new ω6 fatty acids,namely 7.3:1.

T₂ seeds of selected lines transformed with pJP3416-GA7, namely forlines designated 7, 10, 14, 22 and 34 in the Columbia background and forlines designated 18, 21 and 25 in the fad2 mutant background, wereplated onto MS media containing PPT for selection of transgenicseedlings in vitro. Twenty PPT-resistant seedlings for each line weretransferred to soil and grown to maturity after self-fertilisation.These plants were highly likely to be homozygous for the selectablemarker gene, and therefore for at least one T-DNA insertion in thegenome of the plants. T₃ seed from these plants were harvested andanalysed for fatty acid composition in their seedoil by GC. Thisanalysis revealed that the pJP3416-GA7 construct generated higher levelsof the ω3 LC-PUFA DHA in 13 seeds of the homozygous plants than in thesegregating T₂ seed. Up to about 13.9% DHA was observed in the T3pJP3416-GA7 transformed line designated 22.2 in the Columbia background,increased from about 5.5% in the hemizygous T2 seed, with a sum level ofabout 24.3% of new ω3 fatty acids as a percentage of the total fattyacids in the seed lipid content. New ω6 fatty acids were at a level of1.1% of total fatty acids, representing a very favourable ratio of newω3 fatty acids:new ω6 fatty acids, namely about 22:1. Similarly,transformants in the fad2 mutant background yielded 20.6% as a sum ofnew ω3 fatty acids, including 11.5% DHA, as a percentage of the totalfatty acids in the seed lipid content.

Enzymatic conversion efficiencies for each enzyme step in the pathwayfor production of DHA from oleic acid are shown in Table 4 for the 13seeds with the higher DHA levels. The Δ12-desaturase conversionefficiency in seeds of line 22.2 was 81.6% and the ω3-desaturaseefficiency was 89.1%, both of them remarkably high and indicating thatthese fungal (yeast) enzymes were able to function well in developingseeds. The activities of the other exogenous enzymes in the DHA pathwaywere similarly high for ω3 substrates with the Δ6-desaturase acting at42.2% efficiency, Δ6-elongase at 76.8%, Δ5-desaturase at 95.0%,Δ5-elongase at 88.7% and Δ4-desaturase at 93.3% efficiency. TheΔ6-desaturase activity on the ω6 substrate LA was much lower, with theΔ6-desaturase acting at only 0.7% conversion efficiency on LA. GLA waspresent at a level of only 0.4% and was the only new ω6 product asidefrom 20:2ω6 detected in the T₃ seeds with the highest DHA content.Compiled data from the total seed lipid profiles from independenttransgenic seed are shown in Table 5.

TABLE 4 Conversion efficiencies of the individual enzymatic steps forthe production of DHA from oleic acid, observed in total seed lipid fromtransgenic T₃ Arabidopsis seeds. GA7_ GA7_ GA7_ GA7_ GA7_ GA7_FAD2-GA7_FAD2- GA7_FAD2- T₄ Col_22.2 T₄ Col_22.2 Col_7.2 Col_34.2 Col_10.13Col_22.2 Col_14.19 5.10 21.2 18.14 (mean) best line Omega-6 d12-des73.4% 73.1% 75.7% 81.6% 73.4% 66.6% 78.5% 63.1% 67.6% 82.7% d15-des85.3% 84.4% 86.2% 89.1% 70.2% 87.5% 82.2% 87.6% 81.0% 90.9% d6-des 0.3%0.3% 0.3% 0.7% 0.3% 0.6% 1.0% 0.2% 1.3% 0.7% (d9-elo) 1.7% 1.7% 1.2%1.2% 2.6% 1.1% 2.0% 1.3% 1.6% 1.5% d6-elo d5-des d5-elo d4-des Omega-3d6-des 30.7% 29.3% 28.2% 42.2% 30.2% 38.5% 40.0% 29.2% 41.0% 45.7%(d9-elo) 2.7% 2.7% 2.3% 2.4% 3.0% 2.3% 2.7% 2.9% 2.8% 3.1% d6-elo 75.0%81.1% 79.0% 76.8% 70.9% 79.2% 73.2% 79.1% 77.5% 77.7% d5-des 94.0% 94.6%94.5% 95.0% 97.9% 87.8% 93.3% 91.1% 95.0% 95.8% d5-elo 91.9% 91.7% 93.6%88.7% 89.5% 89.9% 92.2% 91.6% 90.8% 90.2% d4-des 93.2% 93.7% 94.4% 93.3%93.7% 92.5% 95.0% 93.9% 92.2% 90.9%

TABLE 5 Compiled data from the total seed lipid profiles fromindependent transgenic seed. GA7- GA7- GA7-Co1_ GA7-Col_ GA7-Col_GA7-FAD2- GA7-FAD2- GA7-FAD2- T₄ Col_22.2 T₄ Col_22.2 Parameter Col_7.2Col_34.2 10.13 22.2 14.19 25.10 21.2 18.14 (mean ± SD) best line totalw3 (% of 50.0 48.9 51.6 55.8 38.6 47.1 49.4 44.8 54.0 55.9 total FA)total w6 (% of 8.7 9.1 8.3 6.7 16.3 6.7 10.7 6.3 6.7 5.7 total FA) w3/w6ratio 5.75 5.37 6.22 8.33 2.37 7.03 4.62 7.11 8.06 9.81 w6/w3 ratio 0.170.19 0.16 0.12 0.42 0.14 0.22 0.14 0.12 0.10 total novel w3 16.3 15.215.5 24.3 12.5 18.8 20.5 14.0 23.0 26.4 (% of total FA) total novel w61.2 1.2 0.9 1.1 1.5 0.9 1.8 0.7 1.4 1.4 (% of total FA) novel w3/w613.58 12.67 17.22 22.09 8.33 20.89 11.39 20.00 16.43 18.86 ratio novelw6/w3 0.07 0.08 0.06 0.05 0.12 0.05 0.09 0.05 0.06 0.05 ratio OA to EPA14.1% 13.3% 13.4% 21.8% 10.2% 15.0% 16.8% 11.2% 20.4% 24.5% efficiencyOA to DHA 12.0% 11.4% 11.8% 18.0% 8.6% 12.6% 14.8% 9.6% 17.1% 20.1%efficiency LA to EPA 18.9% 18.4% 17.9% 26.9% 14.2% 22.9% 21.8% 18.0%26.2% 29.9% efficiency LA to DHA 16.2% 15.9% 15.7% 22.2% 12.0% 19.1%19.1% 15.5% 21.9% 24.5% efficiency ALA to EPA 22.2% 21.9% 20.7% 30.1%20.2% 26.1% 26.5% 20.5% 29.4% 32.9% efficiency ALA to DHA 19.0% 18.8%18.2% 24.9% 17.1% 21.9% 23.3% 17.6% 24.6% 27.0% efficiency totalsaturates 16.0 14.7 15.4 16.0 16.2 13.4 16.5 12.9 16.0 17.8 total 23.725.8 23.4 19.2 26.5 30.9 21.3 34.3 21.1 18.1 monounsaturates total 58.758.0 59.9 62.5 54.9 53.8 60.1 51.1 60.7 61.6 polyunsaturates total C2019 19.8 16.8 15.9 19.1 21.5 18.2 23.3 18 16.6 total C22 11.4 11 10.815.5 8.6 12.1 13.2 9.9 15.4 17.5 C20/C22 ratio 1.67 1.80 1.56 1.03 2.221.78 1.38 2.35 1.17 0.95

T₃ seeds from the pJP3416-GA7 line 22.2 in the Columbia background,which were progeny from T₂ line 22, were sown directly to soil and thefatty acid composition of mature seed from the resultant T₃ plantsanalysed by GC. The average DHA level of these seeds was 13.3%±1.6(n=10) as a percentage of total fatty acids in the seed lipid. The linewith the highest level of DHA contained 15.1% DHA in the total fattyacids of the seed lipid. The enzymatic conversion efficiencies are shownin Table 4 for each step in the production of DHA from oleic acid.

Southern blot hybridisation analysis was performed. The results showedthat the high-accumulating DHA lines were either single- or double-copyfor the T-DNA from the pJP3416-GA7 construct with the exception oftransgenic line Columbia#22, which had three T-DNA insertions in thegenome of the Arabidopsis plant. The T5 generation seed was alsoanalysed and found to have up to 13.6% DHA in the total seed lipids. TheGA7 construct was found to be stable across multiple generations interms of DHA production capability.

Determination of Oil Content in Transgenic A. thaliana DHA Lines

The oil content of transgenic A. thaliana seeds with various levels ofDHA was determined by GC as described in Example 1. The data are shownin FIG. 4, graphing the oil content (% oil by weight of seed) againstthe DHA content (as a percentage of total fatty acids). Up to 26.5 mg ofDHA per gram of seed was observed (Table 6). The oil content of thetransgenic Arabidopsis seeds was found to be negatively correlated withDHA content. The amount of DHA per weight of seed was greater in thetransformed seeds with a DHA level of about 9% relative to the seedswith about 14% DHA. Subsequent data from species other than Arabidopsishas shown that this negative correlation is more pronounced inArabidopsis than in C. sativa or Brassica species (Example 8 below).

TABLE 6 Proportion and amount of DHA in GA7-transformed Arabidopsisseeds. DHA content DHA content Oil content per weight (% of TFA) (% oilper g seeds) (mg/g seed) GA7/col 22.2-1 14.2 14.89 20.2 GA7/col 22.2-214.3 15.02 20.5 GA7/col 22.2-3 14.0 15.92 21.2 GA7/col 10.15-1 8.7 30.2325.06 GA7/col 10.15-2 8.6 31.25 25.77 GA7/col 10.15-3 8.8 31.70 26.49

Example 3. Stable Expression of a Transgenic DHA Pathway in Camelinasativa Seeds

The binary vector pJP3416-GA7 as described above was introduced into A.tumefaciens strain AGL1 and cells from a culture of the transformedAgrobacterium used to treat C. sativa flowering plants using a floraldip method for transformation (Lu and Kang, 2008). After growth andmaturation of the plants, the T₁ seeds from the treated plants wereharvested, sown onto soil and the resultant plants treated by sprayingwith the herbicide BASTA to select for plants which were transgenic for,and expressing, the bar selectable marker gene present on the T-DNA ofpJP3416-GA7. Surviving T₁ plants which were tolerant to the herbicidewere grown to maturity after allowing them to self-fertilise, and theresultant T₂ seed harvested. Five transgenic plants were obtained, onlythree of which contained the entire T-DNA.

Lipid was extracted from a pool of approximately twenty seeds from eachof the three plants that contained the entire T-DNA. Two of the pooledsamples contained very low, barely detectable levels of DHA, but thethird pool contained about 4.7% DHA. Therefore, lipid was extracted from10 individual T₂ seeds from this plant and the fatty acid compositionanalysed by GC. The fatty acid composition data of the individual seedsfor this transformed line is also shown in Table 7. Compiled data fromthe total seed lipid profiles (Table 7) are shown in Table 8.

DHA was present in six of the 10 individual seeds. The four other seedsdid not have DHA and were presumed to be null segregants which did nothave the T-DNA, based on hemizygosity of the T-DNA insertion in theparental plant. Extracted lipid from the single seed with the highestlevel of DHA had 9.0% DHA while the sum of the percentages for EPA, DPAand DHA was 11.4%.

TABLE 7 Fatty acid composition of total seed lipids from transgenic T₂Camelina saliva seeds transformed with the T-DNA from pJP3416-GA7. Thefatty acid composition is shown for a pooled seed batch (FD5.46) and for10 single seeds ranked (left to right) from highest to lowest DHA. FattyFD5.46 acid pooled # 2 # 4 # 8 # 7 # 9 #1 # 3 # 5 # 6 # 10 14:0 0 0.20.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 16:0 11.6 12.1 12.3 12.1 13.2 12.312.8 11.9 11.4 11.5 11.7 16:1 0.2 0.0 0.1 0.1 0.0 0.2 0.0 0.2 0.2 0.20.2 16:3 0.3 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18:0 3.7 3.3 3.2 3.23.0 3.1 3.2 3.3 3.1 3.2 3.2 18:1 10.8 8.0 8.0 8.6 8.5 9.4 11.0 10.2 8.39.4 8.6 18:1d11 1.7 1.3 1.4 1.4 1.7 1.4 1.5 1.3 1.3 1.3 1.3 18:2 24.718.2 19.5 19.2 18.5 20.1 23.8 32.2 30.3 29.8 31.6 18:3ω3 27.4 26.7 26.627.3 28.9 28.2 27.4 28.3 29.2 29.5 28.2 18:3ω6 0.2 1.4 0.3 0.3 0.4 0.20.5 0.0 ' 0.5 0.4 0.6 20:0 1.6 1.4 1.3 1.4 1.2 1.4 1.4 1.8 2.1 1.9 2.018:4ω3 2.2 6.8 6.4 5.7 7.2 5.7 4.1 0.0 0.0 0.0 0.0 20:1d11 5.3 4.4 4.64.8 3.3 4.1 3.5 4.4 6.1 5.8 5.5 20:1iso 0.4 0.3 0.3 0.3 0.3 0.3 0.0 0.50.6 0.5 0.5 20:2ω6 0.8 0.8 0.9 0.8 0.6 0.8 0.7 1.3 1.5 1.4 1.4 20:3ω30.6 0.8 0.8 0.8 0.7 0.8 0.7 0.6 0.7 0.7 0.6 22:0 0.4 0.5 0.5 0.5 0.4 0.50.5 0.6 0.6 0.6 0.6 20:4ω3 0.2 0.3 0.3 0.3 0.4 0.4 0.5 0.0 0.0 0.0 0.022:1 1.1 1.1 1.2 1.1 0.5 0.9 0.8 1.6 2.2 1.9 2.0 20:5ω3 0.7 1.3 1.6 1.51.6 1.1 1.7 0.0 0.0 0.0 0.1 22:2ω6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.30.2 0.2 22:4ω6 + 0.3 0.2 0.3 0.3 0.0 0.3 0.0 0.4 0.6 0.5 0.5 22:3ω3 24:00.3 0.3 0.3 0.3 0.0 0.3 0.0 0.4 0.4 0.4 0.4 24:1 0.3 0.4 0.4 0.3 0.0 0.30.0 0.5 0.6 0.5 0.5 22:5ω3 0.3 1.1 1.2 1.1 1.1 0.9 0.8 0.0 0.0 0.0 0.022:6ω3 4.7 9.0 8.5 8.3 8.3 7.1 4.9 0.0 0.0 0.0 0.0

TABLE 8 Compiled data from the total seed lipid profiles from transgenicseed as shown in Table 7. Calculations do not include the ‘minor fattyacids’ in Table 7. FD5.46 Parameter pooled # 2 # 4 # 8 # 7 # 9 # 1 # 3 #5 # 6 # 10 total w3 (% of total FA) 36.1 46 45.4 45 48.2 44.2 40.1 28.929.9 30.2 28.9 total w6 (% of total FA) 25.8 20.4 20.7 20.3 19.5 21.1 2533.7 32.6 31.8 33.8 w3/w6 ratio 1.40 2.25 2.19 2.22 2.47 2.09 1.60 0.860.92 0.95 0.86 w6/w3 ratio 0.71 0.44 0.46 0.45 0.40 0.48 0.62 1.17 1.091.05 1.17 total novel w3 8.1 18.5 18 16.9 18.6 15.2 12 0 0 0 0.1 (% oftotal FA) total novel w6 1.1 2.2 1.2 1.1 1 1 1.2 1.5 2.3 2 2.2 (% oftotal FA) novel w3/w6 ratio 7.36 8.41 15.00 15.36 18.60 15.20 10.00 0.05novel w6/w3 ratio 0.14 0.12 0.07 0.07 0.05 0.07 0.10 22.00 OA to EPAefficiency 8.2% 15.6% 15.5% 15.1% 15.1% 12.8% 10.5% 0.0% 0.0% 0.0% 0.1%OA to DHA efficiency 6.7% 12.3% 11.6% 11.5% 11.4% 10.0% 7.0% 0.0% 0.0%0.0% 0.0% LA to EPA efficiency 9.2% 17.2% 17.1% 16.7% 16.2% 13.9% 11.4%0.0% 0.0% 0.0% 0.2% LA to DHA efficiency 7.6% 13.6% 12.9% 12.7% 12.3%10.9% 7.5% 0.0% 0.0% 0.0% 0.0% ALA to EPA efficiency 15.8% 24.8% 24.9%24.2% 22.8% 20.6% 18.5% 0.0% 0.0% 0.0% 0.3% ALA to DHA efficiency 13.0%19.6% 18.7% 18.4% 17.2% 16.1% 12.2% 0.0% 0.0% 0.0% 0.0% total saturates17.6 17.8 17.8 17.6 18 17.8 18.1 18.2 17.7 17.8 18.1 totalmonounsaturates 19.8 15.5 16 16.6 14.3 16.6 16.8 18.7 19.3 19.6 18.6total polyunsaturates 62.5 66.6 66.4 65.6 67.7 65.6 65.1 63 63.1 62.563.2 total C20 9.6 9.3 9.8 9.9 8.1 8.9 8.5 8.6 11 10.3 10.1 total C225.4 10.3 10 9.7 9.4 8.3 5.7 0.6 0.9 0.7 0.7 C20/C22 ratio 1.78 0.90 0.981.02 0.86 1.07 1.49 14.33 12.22 14.71 14.43

Homozygous seed from this line was obtained in the T4 generation. Up to10.3% DHA was produced in event FD5-46-18-110 with an average of 7.3%DHA observed across the entire T4 generation. A subsequent generation(T5) was established to further test the stability of PUFA productionover multiple generations, particularly the DHA. The maximum DHA levelsobserved was found to be stable in the fifth generation, even though thepooled seed DHA content had not stabilised until the T₄ generation dueto the presence of multiple transgenic loci. T₅ seed batches were alsogerminated on MS media in vitro alongside parental C. sativa seed withno obvious difference in germination efficiency or speed observed.Further generations of the transgenic line (T6, T7 generations etc) didnot show any reduction in the seed DHA level. The transgenic plants werefully male and female fertile, and the pollen showed about 100%viability as for the wild-type plants. Analysis of the oil content ofthe seeds having different levels of DHA did not identify a correlationbetween DHA level and oil content, contrary to the correlation seen inArabidopsis thaliana.

In several further transgenic lines, the DHA content of single seedsfrom independent events exceeded 12%. The transgenic:null ratio of theselines was found to be between approximately 3:1 and 15:1. Analysis ofrepresentative fatty acid profiles from the top DHA samples from eachconstruct found only 1.2-1.4% GLA with no other new ω6 PUFA detected. Incontrast, new ω3 PUFA (SDA) ω3 LC-PUFA (ETA, EPA, DPA, DHA) were foundto accumulate to 18.5% with a DHA level of 9.6% of the total fatty acidcontent. Δ6-desaturation was 32% and EPA was 0.8% of the total fattyacid content. The Δ5-elongation efficiency was 93% and Δ6-elongationefficiency was 60%. DHA was detected in the polar seed lipid fraction ofGA7 lines.

It was noted that the segregation ratios observed (˜3:1 to ˜15:1)indicated that one or, at most, two transgenic loci were required toproduce fish oil-like levels of DHA in C. sativa. This had importantimplications for the ease with which the transgenic trait can be bred aswell as for transgene stability.

Homozygous seed was planted out across several glasshouses to generate atotal of over 600 individual plants. Oil was extracted from the seedusing a variety of methods including soxhlet, acetone and hexaneextractions.

¹³C NMR regiospecificity analysis was performed on the transgenic C.sativa seed oil to determine the positional distribution of the ω3LC-PUFA on TAG. An event with approximately equal EPA and DHA wasselected to maximise response for these fatty acids and the ratio ofsn-1,3 to sn-2 was found to be 0.75:0.25 for EPA and 0.86:0.14 for DHAwhere an unbiased distribution would be 0.66:0.33. That is, 75% of theEPA and 86% of the DHA were located at the sn-1,3 position of TAG. Thisindicated that both fatty acids were preferentially located on thesn-1,3 positions in C. sativa TAG although the preference for EPA wasweaker than for DHA. The finding that DHA was predominantly found onsn-1,3 was similar to results previously reported in A. thaliana seed(Petrie et al., 2012).

Since only a small number of independent transgenic lines were obtainedin the transformation experiment described above, further C. sativatransformations were performed using the GA7-modB construct (Example 4).More transformants were obtained and homozygous lines producing inexcess of 20.1% DHA are identified.

Example 4. Modifications to T-DNAs Encoding DHA Pathways in Plant Seeds

In order to improve the DHA production level in B. napus beyond thelevels described in WO2013/185184, the binary vectors pJP3416-GA7-modA,pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modEand pJP3416-GA7-modF were constructed as described in WO2013/185184 andtested in transgenic plants. These binary vectors were variants of thepJP3416-GA7 construct described in Example 2 and were designed tofurther increase the synthesis of DHA in plant seeds, particularly byimproving d6-desaturase and Δ6-elongase functions. SDA had been observedto accumulate in some seed transformed with the GA7 construct due to arelatively low Δ6 elongation efficiency compared to the Δ5-elongase, soamongst other modifications, the two elongase gene positions wereswitched in the T-DNA.

The two elongase coding sequences in pJP3416-GA7 were switched in theirpositions on the T-DNA to yield pJP3416-GA7-modA by first cloning a newP. cordata Δ6-elongase cassette between the SbfI sites of pJP3416-GA7 toreplace the P. cordata Δ5-elongase cassette. This construct was furthermodified by exchanging the FP1 promoter driving the M. pusillaΔ6-desaturase with a conlinin Cnl2 promoter (pLuCnl2) to yieldpJP3416-GA7-modB. This modification was made in an attempt to increasethe Δ6-desaturase expression and thereby enzyme efficiency. It wasthought that the Cnl2 promoter might yield higher expression of thetransgene in B. napus than the truncated napin promoter.

Eight transgenic pJP3416-GA7-modB A. thaliana events and 15 transgenicpJP3416-GA7-modG A. thaliana events were generated. Between 3.4% and7.2% DHA in pooled pJP3416-GA7-modB seed was observed and between 0.6and 4.1% DHA in pooled T2 pJP3416-GA7-modG seed was observed. Several ofthe highest pJP3416-GA7-modB events were sown out on selectable mediaand surviving seedlings taken to the next generation. Seed is beinganalysed for DHA content. Since the pooled T1 seeds representedpopulations that were segregating for the transgenes and included anynull segregants, it is expected that the homozygous seeds from progenyplants would have increased levels of DHA, up to 30% of the total fattyacid content in the seed oil. The other modified constructs were used totransform A. thaliana. Although only a small number of transformed lineswere obtained, none yielded higher levels of DHA than the modBconstruct.

The pJP3416-GA7-modB construct was also used to generate transformed B.napus plants of cultivar Oscar and of a series of breeding linesdesignated NX002, NX003, NX005, NX050, NX052 and NX054. A total of 1558transformed plants were obtained including 77 independent transformedplants (TO) for the Oscar transformation, and 1480 independent plantsfor the breeding lines including 189 for NX005 which is a line having ahigh oleic acid content in its seedoil by virtue of mutations in FAD2genes. The other breeding lines had higher levels of LA and ALA.Transgenic plants which exhibited more than 4 copies of the T-DNA asdetermined by a digital PCR method (Example 1) were discarded; about 25%of the TO plants were discarded by this criterion. About 53% of the TOtransgenic plants had 1 or 2 copies of the T-DNA as determined by thedigital PCR method, 12% had about 3 copies and 24% 4 or more copies.Seed (T1 seed) was harvested from about 450 of the transgenic linesafter self-fertilisation, achieved by bagging the plants duringflowering to avoid out-crossing. T1 seed are harvested from theremainder of the transgenic plants when mature. About 1-2% of the plantlines were either male or female sterile and produced no viable seeds,these TO plants were discarded.

Pools of seed (20 T1 seeds in each pool) were tested for levels of DHAin the pooled seed oil, and lines which showed the highest levels wereselected. In particular, lines having a DHA content of at least 2% ofthe total fatty content in the pooled T1 seeds were selected. About 15%of the transgenic lines were selected in this way; the other 85% werediscarded. Some of these were designated lines CT132-5 (in cultivarOscar), CT133-15, -24, -63, -77, -103, -129 and -130 (in NX005).Selected lines in NX050 included CT136-4, -8, -12, -17, -19, -25, -27,-49 and -51. Twenty seeds from selected lines including CT132.5 and 11seeds from CT133.15 were imbibed and, after two days, oil was extractedfrom a half cotyledon from each of the individual seeds. The other halfcotyledons with embryonic axes were kept and cultured on media tomaintain the specific progeny lines. The fatty acid composition in theoil was determined; the data is shown in Table 9 for CT132.5. The DHAlevel in ten of the 20 seeds analysed was in the range of 7-20% of thetotal fatty acid content as determined by the GC analysis. Other seedshad less than 7% DHA and may have contained a partial (incomplete) copyof the T-DNA from pJP3416-GA7-modB. The transgenic line appeared tocontain multiple transgene insertions that were genetically unlinked.The seeds of transgenic line CT133.15 exhibited DHA levels in the range0-5%. Seeds with no DHA were likely to be null segregants. These dataconfirmed that the modB construct performed well for DHA production incanola seed.

Twenty or 40 individual seeds (T2 seeds) obtained from each of multipleT1 plants, after self-fertilisation, from the selected transformed lineswere tested individually for fatty acid composition. Seeds comprisingDHA at levels greater than 20% were identified (Table 10). Tworepresentative samples, CT136-27-18-2 and CT136-27-18-19 had 21.2% and22.7% DHA, respectively. The total ω3 fatty acid content in these seedswas about 60% as a percentage of the total fatty acid content, and theω6 content was less than 10%. Further sets of 20 or 40 T2 seeds fromeach of the T1 plants were tested for fatty acid composition.Representative data for DHA levels in the total fatty acid content ofseedoil from individual T2 seeds is shown in FIG. 10. Seeds comprisingup to 34.3% DHA were identified, for example in seed CT136-27-47-25(Table 12). The fatty acid composition for seedoil obtained fromCT136-27-47-25 is shown in Table 12. The fatty acid composition included34.3% DHA together with about 1.5% DPA, 0.6% EPA and 0.5% ETA. The SDAlevel was about 7.5%, ALA about 21.9% and LA about 6.9%. The new ω6 PUFAexhibited 1.1% GLA but no detectable ω6-C20 or -C22 LC-PUFA. Totalsaturated fatty acids: 9.6%; monounsaturated fatty acids, 12.5%; totalPUFA, 75.2%; total ω6-PUFA (including LA), 7.2%; total ω3-PUFA, 66.9%;the ratio of total ω6:ω3 fatty acids, 9.3:1; new ω6:new ω3 fatty acids,37:1. The efficiencies of each of the enzymatic steps from oleic acid toDHA were as follows: Δ12-desaturase, 90%; Δ15/ω3-desaturase, 89%;Δ6-desaturase, 67%; Δ6-elongase, 83%; Δ5-desaturase, 99%; Δ5-elongase,98%; Δ4-desaturase, 96%. The overall efficiency of conversion of oleicacid to DHA was about 50%. It was therefore clear that seeds producingDHA in the range of 20.1-35% of the total fatty acid content of theseedoil could be identified and selected, including seeds having between20.1% and 30% DHA or between 30% and 35% DHA in the total fatty acidcontent.

The oil content in some seeds was decreased from about 44% in wild-typeseeds to about 31-39% in some of the DHA producing seeds, but wassimilar to wild-type levels in other DHA producing seeds.

Various transformed plant lines which were producing DHA at levels of atleast 10% in T2 seed are crossed and the F1 progeny selfed in order toproduce F2 progeny which are homozygous for multiple T-DNA insertions.Seedoil from homozygous seed is analysed and up to 30% or 35% of thetotal fatty acid content in the seed oil is DHA.

The TAG in the oil obtained from CT136-27-18-2 and CT136-27-18-19 wasanalysed by ¹³C NMR regiospecificity assay for positional distributionof the DHA on the glycerol backbone of the TAG molecules. The DHA waspreferentially linked at the sn-1,3 position. More than 70%, indeed morethan 90% of the DHA was in the sn-1,3 position.

In several further transgenic lines, the DHA content of single seedsfrom independent events exceeded 12%. The transgenic:null ratio of theselines was found to be approximately 3:1, corresponding to a singletransgenic locus, or 15:1, corresponding to two transgenic loci.Analysis of representative fatty acid profiles from the samples fromeach construct with the highest levels of DHA found only 1.2-1.4% GLAwith no other new ω6 PUFA detected. In contrast, new ω3 PUFA (SDA) andω3 LC-PUFA (ETA, EPA, DPA, DHA) accumulated to a sum of 25.8% for themodF construct and 21.9% for the modG construct compared to 18.5% forthe GA7-transformed seed. The DHA levels in the oil from these seedswere 9.6%, 12.4% and 11.5%, respectively. Δ6-desaturation was found tobe lower in the GA7-transformed seeds than the modF- andmodG-transformed seeds (32% vs 47% and 43%) and this resulted in areduction of ALA in the modF and modG seeds relative to GA7. Anothernoteworthy difference was the accumulation of EPA in the modF seed (3.3%vs 0.8% in the other two transgenic seeds) and this was reflected in thereduced Δ5-elongation observed in modF (80%) seed relative to GA7 andmodG seeds (93% and 94%). There was a slight increase in Δ6-elongationin these seeds (66% vs 60% and 61%) although the amount of SDA actuallyincreased due to the slightly more active ω6-desaturation. DHA wasdetected in the polar seed lipid fraction of GA7 lines.

The fatty acid composition was analysed of the lipid in the T1 seed of70 independent transgenic plants of the B. napus breeding line NX54transformed with the T-DNA of the modB construct. It was observed thatone of these transgenic plants produced seed having DPA but no DHA inthe seedoil. The T1 seed of this line (CT-137-2) produced about 4% DPAwithout any detectable DHA in the T1 pooled seed. The inventorsconcluded that this was caused by inactivation of the Δ4-desaturase genein that particular inserted T-DNA, perhaps through a spontaneousmutation. Around 50 T1 seeds from this transgenic line were germinatedand one emerged cotyledon from each analysed for fatty acid compositionin the remaining oil. Selected seedlings exhibiting more than 5% DPAwere then grown to maturity and T2 seed harvested. Pooled seed fattyacid compositions are shown in Table 11, more than 7% DPA was observedin these lines.

Whilst the focus of this experiment was the demonstration of DHAproduction in an oilseed crop species, the differences noted above werealso interesting from a construct design perspective. First, switchingthe Δ6- and Δ5-elongase coding region locations in the modF constructresulted in the intended profile change with more EPA accumulated due tolower Δ5-elongation. A concomitant increase in Δ6-elongation wasobserved but this did not result in lower SDA levels. This was due to anincrease in Δ6-desaturation in the modF transformed seed, caused byadding an extra M. pusilla Δ6-desaturase expression cassette as well asby replacing the truncated napin promoter (FP1) with a more highlyactive flax conlinin2 promoter. The somewhat lower increase inΔ6-desaturation observed with the modG construct was caused bycapitalising on the highly expressed Δ5-elongase cassette in GA7.Switching the positions of the Δ6-desaturase and Δ5-elongase codingregions resulted in greater Δ6-desaturation. Δ5-elongase activity wasnot reduced in this instance due to the replacement of the FP1 promoterwith the Cnl2 promoter.

These data confirmed that the modB, modF and modG constructs performedwell for DHA production in Camelina seed, as for Arabidopsis and canola.

The inventors considered that, in general, the efficiency ofrate-limiting enzyme activities in the DHA pathway can be greater inmulticopy T-DNA transformants compared to single-copy T-DNAtransformants, or can be increased by inserting into the T-DNA multiplegenes encoding the enzyme which might be limiting in the pathway.Evidence for the possible importance of multi-copy transformants wasseen in the Arabidopsis seeds transformed with the GA7 construct(Example 2), where the highest yielding DHA event had three T-DNAsinserted into the host genome. The multiple genes can be identical, orpreferably are different variants that encode the same polypeptide, orare under the control of different promoters which have overlappingexpression patterns. For example, increased expression could be achievedby expression of multiple Δ6-desaturase coding regions, even where thesame protein is produced. In pJP3416-GA7-modF and pJP3416-GA7-modC, forinstance, two versions of the M. pusilla Δ6-desaturase were present andexpressed by different promoters. The coding sequences had differentcodon usage and therefore different nucleotide sequences, to reducepotential silencing or co-suppression effects but resulting in theproduction of the same protein.

TABLE 9 Fatty acid profiles of half cotyledons of germinating T1transgenic B. napus seeds containing the modB construct. Up to 18.1% DHAwas observed with numerous samples containing greater than 10% DHA. Seed14:0 16:0 16:1d3? 16:1 16:3 18:0 18:1 18:1d11 18:2 18:3n6 18:3n3 20:018:4n3 1 0.1 4.2 0.1 0.1 0.2 1.8 29.9 2.5 9.9 0.1 38.4 0.5 0.8 2 0.1 4.70.1 0.1 0.2 4.0 23.0 2.3 7.4 0.3 29.3 1.0 4.3 3 0.1 3.7 0.2 0.1 0.2 1.855.1 1.9 4.7 0.2 15.2 0.8 1.8 4 0.1 4.6 0.2 0.2 0.2 2.9 22.1 1.8 6.6 0.426.5 1.0 7.2 5 0.1 4.0 0.1 0.1 0.2 1.7 27.4 2.1 8.1 0.3 26.4 0.6 2.8 60.1 3.5 0.1 0.1 0.2 1.6 59.8 2.0 4.3 0.1 18.5 0.6 0.5 7 0.1 6.0 0.3 0.30.3 1.7 16.6 2.6 23.9 1.0 23.2 0.6 5.4 8 0.1 4.9 0.1 0.1 0.2 2.7 12.91.4 11.7 0.3 34.3 0.9 5.0 9 0.1 3.9 0.1 0.1 0.1 2.4 41.6 1.7 21.5 0.023.4 0.7 0.0 10 0.1 3.7 0.2 0.1 0.1 2.1 30.9 1.7 19.2 0.4 23.6 0.7 2.111 0.1 5.7 0.4 0.3 0.2 3.8 41.2 2.4 26.7 2.1 7.2 1.3 0.3 12 0.1 4.6 0.00.1 0.2 2.4 25.5 1.7 16.1 0.3 28.9 0.8 3.9 13 0.1 4.3 0.1 0.1 0.1 4.219.4 1.6 9.2 0.1 45.5 1.0 0.2 14 0.1 6.3 0.2 0.2 0.2 4.0 10.5 2.3 8.40.3 31.1 1.3 3.9 15 0.1 5.1 0.1 0.2 0.2 3.3 16.8 2.4 11.2 0.3 28.8 1.04.5 16 0.1 4.4 0.1 0.1 0.2 4.0 16.2 1.5 11.6 0.2 33.5 0.9 2.8 17 0.2 7.20.2 0.2 0.2 4.9 15.0 2.1 8.9 0.3 25.9 1.4 5.1 18 0.1 4.0 0.1 0.1 0.2 2.364.8 1.2 7.2 0.1 12.5 1.0 3.5 19 0.1 3.9 0.1 0.1 0.2 4.6 36.9 1.7 7.10.2 28.6 1.2 1.8 20 0.1 4.8 0.1 0.1 0.2 6.0 18.5 1.2 12.8 0.2 34.8 1.42.4 Seed C20:1d11 20:1d13 C20:2n6 C20:3n3 C22:0 20:4n3 20:5n3 22:3n3C24:0 C24:1 22:5n3 C22:6n3 1 1.0 0.0 0.1 2.1 0.3 2.8 0.3 0.1 0.2 0.2 0.23.9 2 1.1 0.0 0.1 1.9 0.4 6.9 1.0 0.0 0.3 0.1 1.7 9.5 3 1.4 0.0 0.1 0.30.5 11.3 0.0 0.0 0.3 0.2 0.0 0.0 4 1.0 0.0 0.1 0.8 0.5 11.2 1.9 0.0 0.20.2 1.7 8.7 5 1.0 0.0 0.1 1.5 0.3 7.6 1.5 0.0 0.1 0.1 1.8 12.2 6 1.3 0.00.0 0.7 0.3 6.0 0.0 0.0 0.2 0.1 0.0 0.0 7 0.8 0.0 0.2 0.6 0.4 2.6 1.10.0 0.3 0.3 1.7 9.9 8 0.9 0.0 0.2 2.4 0.5 4.1 1.3 0.0 0.2 0.2 1.8 13.8 91.2 0.0 0.1 2.2 0.4 0.0 0.0 0.1 0.3 0.2 0.0 0.0 10 1.1 0.0 0.1 1.5 0.43.6 0.6 0.0 0.2 0.1 0.7 6.9 11 1.2 0.0 0.2 0.3 0.8 4.8 0.0 0.0 0.6 0.30.0 0.0 12 1.1 0.0 0.1 1.9 0.4 3.9 0.6 0.0 0.2 0.0 1.1 6.2 13 1.1 0.00.1 5.2 0.4 2.6 0.3 0.2 0.2 0.1 0.4 3.4 14 0.8 0.0 0.1 2.3 0.6 4.6 1.80.1 0.3 0.2 2.5 18.1 15 0.9 0.0 0.1 2.1 0.6 3.2 1.5 0.1 0.3 0.1 1.8 15.116 1.1 0.0 0.2 3.7 0.4 4.6 0.7 0.1 0.3 0.1 1.3 12.1 17 0.9 0.0 0.0 1.60.8 4.9 2.1 0.0 0.6 0.3 2.2 15.0 18 1.5 0.0 0.1 0.0 0.7 0.0 0.0 0.0 0.50.2 0.0 0.0 19 1.2 0.0 0.1 1.4 0.5 4.3 0.4 0.0 0.4 0.1 0.8 4.3 20 1.10.0 0.1 3.4 0.6 3.2 0.4 0.1 0.3 0.1 0.7 7.6

TABLE 10 Fatty acid profiles of T2 transgenic B. napus seeds containingthe modB construct. Sample (T2 seed) C14:0 C16:0 C18:0 C18:1 C18:1d11C18:2 C18:3n6 C18:3n3 18:4n3 C20:1d11 CT136-27-18-1 0.1 5.0 2.6 25.4 3.66.7 0.2 37.5 1.4 1.0 CT136-27-18-2 0.2 7.1 2.8 16.9 4.3 5.5 0.4 29.1 5.40.8 CT136-27-18-3 0.1 5.4 2.5 26.5 3.8 6.4 0.4 26.4 4.7 1.0CT136-27-18-4 0.1 5.3 2.4 34.7 4.0 5.9 0.3 30.3 1.3 1.1 CT136-27-18-50.1 4.8 2.7 34.5 3.8 5.6 0.3 23.5 3.9 1.2 CT136-27-18-6 0.1 5.0 2.1 54.33.8 5.7 0.2 18.2 0.6 1.5 CT136-27-18-7 0.1 5.3 2.1 43.8 4.2 5.6 0.4 18.32.2 1.3 CT136-27-18-8 0.1 5.4 2.7 25.8 4.1 6.7 0.4 26.6 5.7 1.0CT136-27-18-9 0.1 4.6 1.6 53.8 3.7 17.5 0.5 9.2 0.5 1.6 CT136-27-18-100.1 4.8 2.4 44.1 3.7 5.4 0.4 19.1 2.3 1.1 CT136-27-18-11 0.1 5.1 2.248.3 4.1 10.9 0.7 12.5 1.2 1.3 CT136-27-18-12 0.1 5.3 2.7 23.3 3.7 6.00.4 27.9 4.9 0.9 CT136-27-18-13 0.1 5.5 3.4 30.7 5.6 5.1 0.4 23.1 3.51.1 CT136-27-18-14 0.1 5.4 2.3 23.9 3.5 6.0 0.4 30.1 3.7 1.0CT136-27-18-15 0.1 5.0 2.3 45.4 4.0 5.3 0.4 16.2 2.3 1.2 CT136-27-18-160.1 4.8 2.7 37.9 4.1 6.2 0.4 22.0 2.4 1.0 CT136-27-18-17 0.1 4.5 2.338.8 3.3 7.6 0.3 26.8 0.9 1.4 CT136-27-18-18 0.1 5.1 2.3 29.0 3.6 5.70.4 26.5 3.8 1.1 CT136-27-18-19 0.1 5.8 2.3 19.7 4.2 6.7 0.7 23.7 7.70.9 CT136-27-18-20 0.1 5.7 2.9 23.2 4.0 5.6 0.3 35.8 2.4 1.0 Total TotalTotal Ratio PUFA Sample ω3 ω6 ω6 to content (T2 seed) C20:2n6 C20:3n320:4n3 20:5n3 22:5n3 C22:6n3 (%) (%) ω3 (%) CT136-27-18-1 0.1 2.1 0.80.4 0.9 10.2 53.4 7.1 0.13 60.5 CT136-27-18-2 0.1 1.2 0.5 0.5 1.9 21.259.8 6.1 0.10 66.0 CT136-27-18-3 0.1 0.7 1.1 0.6 1.2 17.3 52.0 6.9 0.1358.9 CT136-27-18-4 0.1 1.1 1.5 0.3 0.4 9.3 44.4 6.3 0.14 50.7CT136-27-18-5 0.1 0.7 1.1 0.5 1.1 14.2 45.1 6.0 0.13 51.1 CT136-27-18-60.1 1.1 0.7 0.1 0.2 4.4 25.5 6.1 0.24 31.5 CT136-27-18-7 0.2 0.6 1.5 0.40.5 11.6 35.2 6.2 0.18 41.4 CT136-27-18-8 0.1 0.6 1.3 0.6 1.2 15.8 51.97.1 0.14 59.0 CT136-27-18-9 0.3 0.6 0.4 0.1 0.1 3.7 14.5 18.3 1.26 32.8CT136-27-18-10 0.1 0.6 1.5 0.5 0.8 11.4 36.1 5.9 0.16 42.0CT136-27-18-11 0.2 0.5 1.5 0.3 0.3 9.1 25.3 11.8 0.47 37.1CT136-27-18-12 0.1 0.7 1.3 0.8 1.5 18.5 55.7 6.6 0.12 62.2CT136-27-18-13 0.1 1.2 1.1 0.6 1.2 14.9 45.8 5.5 0.12 51.3CT136-27-18-14 0.1 1.0 0.7 0.6 1.2 18.2 55.5 6.6 0.12 62.1CT136-27-18-15 0.1 0.5 1.9 0.6 0.7 12.3 34.4 5.8 0.17 40.3CT136-27-18-16 0.1 0.7 1.4 0.5 0.8 13.1 41.0 6.7 0.16 47.7CT136-27-18-17 0.2 1.6 0.9 0.2 0.7 8.6 39.9 8.0 0.20 47.9 CT136-27-18-180.2 0.8 0.8 0.6 1.0 17.4 50.8 6.3 0.12 57.1 CT136-27-18-19 0.1 0.4 0.70.6 1.7 22.7 57.6 7.5 0.13 65.1 CT136-27-18-20 0.1 1.3 1.1 0.5 1.0 13.055.1 6.1 0.11 61.2 ARA (C20:4ω6) was not detected in any of the samples.The samples also contained about 0.2% or 0.3% C16:1, about 0.1 to 0.3%C16:3, between about 0.7% and 1.0% C20:0, about 0.3% C22:0, and somesamples contained brace levels (<0.1%) of C20:1Δ13, C22:3ω3, C24:0 andC24:1

TABLE 11 Fatty acid composition of the lipid in T2 transgenic B. napusseeds transformed with the T-DNA of the modB construct, with a presumedmutaton in the Δ4-desaturase gene. The lipids also contained about 0.1%14:0, 0.2% 16:3, 0.2-0.4% GLA, 0.1% 20:1Δ13, 0.3-0.4% 22:0, and 16:2 and22:1 were not detected. 16:0 16:1ω13t 16:1 18:0 18:1 18:1d11 18:2 18:3n320:0 18:4n3 20:1d11 20:2n6 CT-137-2-34 5.3 0.0 0.2 3.7 26.8 3.1 12.429.1 0.8 2.5 0.8 0.1 CT-137-2-38 5.3 0.0 0.2 4.2 24.4 3.0 12.6 29.4 0.92.5 0.8 0.1 CT-137-2-48 5.0 0.0 0.2 4.2 24.1 3.1 11.9 31.0 0.9 2.4 0.90.1 CT-137-2-51 5.7 0.0 0.2 4.6 22.3 3.4 12.3 34.5 1.0 2.0 0.8 0.1CT-137-2-59 5.4 0.0 0.2 3.9 25.7 3.4 12.9 27.8 0.9 2.6 0.8 0.1 20:3n620:4n6 20:3n3 20:4n3 20:5n3 20:2n6 20:3n3 24:0 24:1 20:5n3 20:6n3CT-137-2-34 0.0 0.0 1.1 1.7 0.8 0.0 0.1 0.1 0.1 10.0 0.0 CT-137-2-38 0.00.0 1.3 2.2 0.9 0.0 0.1 0.2 0.1 10.8 0.0 CT-137-2-48 0.0 0.0 1.5 2.0 1.00.0 0.1 0.1 0.1 10.5 0.0 CT-137-2-51 0.0 0.0 1.9 1.2 0.5 0.0 0.1 0.2 0.27.9 0.0 CT-137-2-59 0.0 0.0 1.0 1.9 0.9 0.0 0.1 0.2 0.1 11.0 0.0

TABLE 12 Fatty acid composition of seedoil from T2 seed of B. napustransformed with the T-DNA from GA7-modB. C16:0 C18:0 C18:1n9 C18:1n7C18:2n6 C18:3n6 C18:3n3 C20:0 C18:4n3 6.3 2.4 8.4 3.1 6.9 1.1 21.9 0.77.5 C20:1n9c C20:2n6 + C21:0 C20:3n3 C20:4n3 C20:5n3 C22:5n6 C22:5n3C22:6n3 0.7 0.1 0.5 0.5 0.6 0.2 1.5 34.3 The seedoil samples alsocontained 0.1% C14:0; 0.2% C16:1; 0.1% C20:3ω6; no C22:1 and C22:2ω6;0.1% C24:0 and 0.2% C24:1, 2.6% other fatty acids.

Example 5. Analysis of TAG from Transgenic A. thaliana Seeds ProducingDHA

The positional distribution of DHA on the TAG from the transformed A.thaliana seed was determined by NMR. Total lipid was extracted fromapproximately 200 mg of seed by first crushing them under hexane beforetransferring the crushed seed to a glass tube containing 10 mL hexane.The tube was warmed at approximately 55° C. in a water bath and thenvortexed and centrifuged. The hexane solution was removed and theprocedure repeated with a further 4×10 mL. The extracts were combined,concentrated by rotary evaporation and the TAG in the extracted lipidpurified away from polar lipids by passage through a short silica columnusing 20 mL of 7% diethyl ether in hexane. Acyl group positionaldistributions on the purified TAG were determined quantitatively aspreviously described (Petrie et al., 2010a and b).

The analysis showed that the majority of the DHA in the total seed oilwas located at the sn-1/3 positions of TAG with little found at the sn-2position (FIG. 5). This was in contrast to TAG from ARA producing seedswhich demonstrated that 50% of the ARA (20:4^(Δ5,8,11,14)) was locatedat the sn-2 position of transgenic canola oil whereas only 33% would beexpected in a random distribution (Petrie et al., 2012).

The total lipid from transgenic A. thaliana seeds was also analysed bytriple quadrupole LC-MS to determine the major DHA-containingtriacylglycerol (TAG) species (FIG. 6). The most abundant DHA-containingTAG species was found to be DHA-1.8:3-18:3 (TAG 58:12; nomenclature notdescriptive of positional distribution) with the second-most abundantbeing DHA-18:3-18:2 (TAG 58:11). Tri-DHA TAG (TAG 66:18) was observed intotal seed oil, albeit at low but detectable levels. Other majorDHA-containing TAG species included DHA-34:3 (TAG 56:9), DHA-36:3 (TAG58:9), DHA-36:4 (TAG 58:10), DHA-36:7 (TAG 58:13) and DHA-38:4 (TAG60:10). The identities of the two major DHA-containing TAG were furtherconfirmed by Q-TOF MS/MS.

Example 6. Assaying Sterol Content and Composition in Oils

The phytosterols from 12 vegetable oil samples purchased from commercialsources in Australia were characterised by GC and GC-MS analysis as0-trimethylsilyl ether (OTMSi-ether) derivatives as described inExample 1. Sterols were identified by retention data, interpretation ofmass spectra and comparison with literature and laboratory standard massspectral data. The sterols were quantified by use of a5β(H)-Cholan-24-ol internal standard. The basic phytosterol structureand the chemical structures of some of the identified sterols are shownin FIG. 7 and Table 13.

The vegetable oils analysed were from: sesame (Sesamum indicum), olive(Olea europaea), sunflower (Helianthus annus), castor (Ricinuscommunis), canola (Brassica napus), safflower (Carthamus tinctorius),peanut (Arachis hypogaea), flax (Linum usitatissimum) and soybean(Glycine max). In decreasing relative abundance, across all of the oilsamples, the major phytosterols were: β-sitosterol (range 28-55% oftotal sterol content), Δ5-avenasterol (isofucosterol) (3-24%),campesterol (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%)and Δ7-avenasterol (0.1-5%). Several other minor sterols wereidentified, these were: cholesterol, brassicasterol, chalinasterol,campestanol and eburicol. Four C29:2 and two C30:2 sterols were alsodetected, but further research is required to complete identification ofthese minor components. In addition, several other unidentified sterolswere present in some of the oils but due to their very low abundance,the mass spectra were not intense enough to enable identification oftheir structures.

TABLE 13 IUPAC/systematic names of identified sterols. Sterol No. Commonname(s) IUPAC/Systematic name 1 cholesterol cholest-5-en-3β-ol 2brassicasterol 24-methylcholesta-5,22E-dien-3β-ol 3 chalinasterol/24-methylcholesta-5,24(28)E-dien- 24-methylene 3β-ol cholesterol 4campesterol/ 24-methylcholest-5-en-3β-ol 24-methylcholesterol 5campestanol/ 24-methylcholestan-3β-ol 24-methylcholestanol 7Δ5-stigmasterol 24-ethylcholesta-5,22E-dien-3β-ol 9 ergost-7-en-3β-ol24-methylcholest-7-en-3β-ol 11 eburicol4,4,14-trimthylergosta-8,24(28)-dien- 3β-ol 12 β-sitosterol/24-ethylcholest-5-en-3β-ol 24-ethylcholesterol 13 D5-avenasterol/24-ethylcholesta-5,24(28)Z-dien- isofucosterol 3β-ol 19 D7-stigmasterol/24-ethylcholest-7-en-3β-ol stigmast-7-en-3b-ol 20 D7-avenasterol24-ethylcholesta 7,24(28)-dien-3β-ol

The sterol contents expressed as mg/g of oil in decreasing amount were:canola oil (6.8 mg/g), sesame oil (5.8 mg/g), flax oil (4.8-5.2 mg/g),sunflower oil (3.7-4.1 mg/g), peanut oil (3.2 mg/g), safflower oil (3.0mg/g), soybean oil (3.0 mg/g), olive oil (2.4 mg/g), castor oil (1.9mg/g). The % sterol compositions and total sterol content are presentedin Table 14.

Among all the seed oil samples, the major phytosterol was generallyβ-sitosterol (range 30-57% of total sterol content). There was a widerange amongst the oils in the proportions of the other major sterols:campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%),Δ7-stigmasterol (1-18%). Oils from different species had a differentsterol profile with some having quite distinctive profiles. In the caseof canola oil, it had the highest proportion of campesterol (33.6%),while the other species samples generally had lower levels, e.g. up to17% in peanut oil. Safflower oil had a relatively high proportion ofΔ7-stigmasterol (18%), while this sterol was usually low in the otherspecies oils, up to 9% in sunflower oil. Because they were distinctivefor each species, sterol profiles can therefore be used to help in theidentification of specific vegetable or plant oils and to check theirgenuineness or adulteration with other oils.

Two samples each of sunflower and safflower were compared, in each caseone was produced by cold pressing of seeds and unrefined, while theother was not cold-pressed and refined. Although some differences wereobserved, the two sources of oils had similar sterol compositions andtotal sterol contents, suggesting that processing and refining hadlittle effect on these two parameters. The sterol content among thesamples varied three-fold and ranged from 1.9 mg/g to 6.8 mg/g. Canolaoil had the highest and castor oil the lowest sterol content.

Example 7. Increasing Accumulation of DHA at the sn-2 TAG Position

The present inventors considered that DHA and/or DPA accumulation at thesn-2 position in TAG could be increased by co-expressing an1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) together with theDHA or DPA biosynthesis pathway such as conferred by the GA7 constructor its variants. Preferred LPAATs are those which can act onpolyunsaturated C22 fatty acyl-CoA as substrate, preferably DHA-CoAand/or DPA-CoA, resulting in increased insertion of the polyunsaturatedC22 chain at the sn-2 position of LPA to form PA, relative to theendogenous LPAAT. Cytoplasmic LPAAT enzymes often display variedsubstrate preferences, particularly where the species synthesises andaccumulates unusual fatty acids in TAG. A LPAAT2 from Limnanthesdouglasii was shown to use erucoyl-CoA (C22:1-CoA) as a substrate for PAsynthesis, in contrast to an LPAAT1 from the same species that could notutilise the C22 substrate (Brown et al., 2002).

TABLE 14 Sterol content and composition of assayed plant oils. Sun- Saf-Sterol flower flower Sterol common Sun- cold- Saf- cold- Flax Flax Soy-number* name Sesame Olive flower pressed Castor Canola flower pressedPeanut (linseed) (linseed) bean 1 cholestercl 0.2 0.8 0.2 0.0 0.1 0.30.2 0.1 0.2 0.4 0.4 0.2 2 brassicasterol 0.1 0.0 0.0 0.0 0.3 0.1 0.0 0.00.0 0.2 0.2 0.0 3 chalinasterol/ 1.5 0.1 0.3 0.1 1.1 2.4 0.2 0.1 0.9 1.51.4 0.8 24-methylene cholesterol 4 campesterol/24- 16.2 2.4 7.4 7.9 8.433.6 12.1 8.5 17.4 15.7 14.4 16.9 methylcholesterol 5 campestarol/24-0.7 0.3 0.3 0.1 0.9 0.2 0.8 0.8 0.3 0.2 0.2 0.7 methylcholestanol 6C29:2* 0.0 0.0 0.1 0.2 0.0 0.1 0.5 0.5 0.0 1.2 1.3 0.1 7 Δ5-stigmasterol6.4 1.2 7.4 7.2 18.6 0.7 7.0 4.6 6.9 5.1 5.8 17.6 8 unknown 0.5 1.3 0.70.6 0.8 0.7 0.7 1.3 0.4 0.7 0.6 1.3 9 ergost-7-en-3β-ol 0.1 0.1 1.9 1.80.2 0.4 2.7 4.0 1.4 1.4 1.4 1.0 10 unknown 0.0 1.3 0.9 0.8 1.2 0.9 1.80.7 1.2 0.7 0.5 0.7 11 eburicol 1.6 1.8 4.1 4.4 1.5 1.0 1.9 2.9 1.2 3.53.3 0.9 12 β-sitosterol/24- 55.3 45.6 43.9 43.6 37.7 50.8 4.0.2 35.157.2 29.9 28.4 40.2 ethylcholesterol 13 Δ5-avenasterol/ 8.6 16.9 7.2 4.119.3 4.4 7.3 6.3 5.3 23.0 24.2 3.3 isofucosterol 14 triterpeno d alcohol0.0 2.4 0.9 1.1 0.0 0.0 1.6 1.9 0.0 0.0 0.0 0.9 15 triterpeno d alcohol0.0 0.0 0.7 0.6 0.0 0.0 2.8 1.8 0.0 0.0 0.3 0.0 16 C29:2* 0.0 0.5 0.70.7 1.5 1.2 2.8 1.9 2.0 1.0 0.7 0.5 17 C29:2* 1.0 0.9 2.3 2.4 0.6 0.41.3 1.9 0.9 1.0 1.0 1.0 18 C30:2* 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.00.0 0.0 0.0 19 Δ7-stigmasterol/ 2.2 7.1 9.3 10.9 2.3 0.9 10.5 18.3 1.17.9 8.7 5.6 stigmast- 7-en-3β-ol 20 Δ7-avenasterol 1.3 0.1 4.0 3.6 0.60.2 2.0 4.7 0.7 0.4 0.4 0.6 21 unknown 0.7 7.1 0.9 0.8 0.0 0.4 0.3 0.40.0 3.0 3.6 0.0 22 unknown 0.3 0.0 0.3 0.9 0.0 0.0 1.2 1.3 0.2 0.1 0.00.3 23 unknown 0.2 0.2 0.3 0.3 0.2 0.1 0.3 0.2 0.2 0.1 0.2 0.5 24unknown 0.0 3.1 0.9 1.3 0.6 0.4 0.2 0.4 0.7 1.7 1.9 0.8 25 unknown 0.90.4 0.3 0.5 0.3 0.1 0.5 0.7 0.3 0.1 0.1 0.6 26 C30:2 2.2 6.0 4.6 5.7 1.40.6 1.0 1.2 1.2 1.2 1.1 5.2 27 unknown 0.0 0.4 0.4 0.3 0.3 0.2 0.1 0.20.3 0.1 0.0 0.3 Sum 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0 100.0 Total sterol 5.8 2.4 4.1 3.7 1.9 6.8 3.2 3.0 3.24.8 5.2 3.0 (mg/g oil) C29:2* and and C30:2* denotes a C29 sterol withtwo double bonds and a C30 sterol with two double bonds, respectively

Known LPAATs were considered and a number were selected for testing,including some which were not expected to increase DHA incorporation atthe sn-2 position, as controls. The known LPAATs included: Arabidopsisthaliana LPAAT2: (SEQ ID NO: 40, Accession No. ABG48392, Kim et al.,2005), Limnanthes alba LPAAT (SEQ ID NO: 41, Accession No. AAC49185,Lassner et al., 1995), Saccharomyces cerevisiae Slc1p (SEQ ID NO: 42,Accession No. NP_010231, Zou et al., 1997), Mortierella alpina LPAAT1(SEQ ID NO: 44, Accession No. AED33305; U.S. Pat. No. 7,879,591) andBrassica napus LPAATs (SEQ ID NO: 45 and SEQ ID NO:46, Accession NosADC97479 and ADC97478 respectively).

The Arabidopsis LPAAT2 (also designated LPAT2) is art endoplasmicreticulum-localised enzyme shown to have activity on C16 and C18substrates, however activity on C20 or C22 substrates was not tested(Kim et al., 2005). Limnanthes alba LPAAT2 was demonstrated to insert aC22:1 acyl chain into the sn-2 position of PA, although the ability touse DHA or DPA as a substrate was not tested (Lassner et al., 1995). Theselected S. cerevisiae LPAAT Slc1p was shown to have activity using22:1-CoA in addition to 18:1-CoA as substrates, indicating a broadsubstrate specificity with respect to chain length (Zou et al., 1997).Again, DHA-CoA, DPA-CoA and other LC-PUFAs were not tested assubstrates. The Mortierella LPAAT had previously been shown to haveactivity on EPA and DHA fatty acid substrates in transgenic Yarrowialipolytica (U.S. Pat. No. 7,879,591) but its activity in plant cells wasunknown.

Additional LPAATs were identified by the inventors. Micromonas pusillais a microalga that produces and accumulates DHA in its oil, althoughthe positional distribution of the DHA on TAG in this species has notbeen confirmed, The Micromonas pusilla LPAAT (SEQ ID NO: 43, AccessionNo. XP_002501997) was identified by searching the Micromonas pusillagenomic sequence using the Arabidopsis LPAAT2 as a BLAST query sequence.Several candidate sequences emerged and the sequence XP_002501997 wassynthesised for testing on C22 LC-PUFA. The Ricinus communis LPAAT wasannotated as a putative LPAAT in the castor genome sequence (Chan etal., 2010). Four candidate LPAATs from the castor genome weresynthesised and tested in crude leaf lysates of infiltrated N.benthamiana leaf tissue. The candidate sequence described here showedLPAAT activity.

A number of candidate LPAATs were aligned with known LPAATs on aphylogenetic tree (FIG. 8). It was noted that the putative MicromonasLPAAT did not cluster with the putative C22 LPAATs but was a divergentsequence.

As an initial test of various LPAATs for their ability to use DHA-CoA assubstrate, chimeric genetic constructs were made for constitutiveexpression of exogenous LPAATs in N. benthamiana leaves, each under thecontrol of the 35S promoter, as follows: 35S:Arath-LPAAT2 (ArabidopsisER LPAAT); 35S:Limal-LPAAT (Limnanthes alba LPAAT); 35S:Sacce-Slc1p (S.cerevisiae LPAAT); 35S:Micpu-LPAAT (Micromonas pusilla LPAAT);35S:Moral-LPAAT1 (Mortierella alpina LPAAT); 35S:Brana-LPAAT1.13(Brassica napus LPAAT1.13); 35 S:Brana-LPAAT1.5 (Brassica napusLPAAT1.5). A 35S:p19 construct lacking an exogenous LPAAT was used as acontrol in the experiment. Each of these constructs was introduced viaAgrobacterium into N. benthamiana leaves as described in Example 1, and5 days after infiltration, the treated leaf zones were excised andground to make leaf lysates. Each lysate included the exogenous LPAAT aswell as the endogenous enzymes for synthesizing LPA. In vitro reactionswere set up by separately adding ¹⁴C-labelled-OA and -DHA to thelysates. Reactions were incubated at 25° C. and the level ofincorporation of the ¹⁴C labelled fatty acids into PA determined by TLC.The ability of each LPAAT to use DHA relative to ARA and the C18 fattyacids were assessed. The meadowfoam (Limnanthes alba), Mortierella andSaccharomyces LPAATs were found to have activity on DHA substrate, withradiolabelled PA appearing for these but not the other LPAATs. AllLPAATs were confirmed active by the oleic acid control feed.

To test LPAAT activity in seeds, several of the protein coding sequencesor LPAATs were inserted into a binary vector under the control of aconlinin (pLuCn11) promoter. The resultant genetic constructs containingthe chimeric genes, Cnl1:Arath-LPAAT (negative control),Cnl1:Limal-LPAAT, Cnl:Sacce-Slc1p, and Cnl1:Moral-LPAAT, respectively,are then used to transform A. thaliana plants producing DHA in theirseed to generate stable transformants expressing the LPAATs and thetransgenic DHA pathway in a seed-specific manner to test whether therewould be an increased incorporation of DHA at the sn-2 position of TAG.The constructs are also used to transform B. napus and C. sativa plantsthat already contain the GA7 construct and variants thereof (Examples 2to 4) to generate progeny carrying both the parental and LPAAT geneticconstructs. Increased incorporation of DHA at the sn-2 position of TAGis tested relative to the incorporation in plants lacking the LPAATencoding transgenes. Oil content is also improved in the seeds,particularly for seeds producing higher levels of DHA, counteracting thetrend seen in Arabidopsis seed as described in Example 2.

The 35S:Moral-LPAAT1 construct was used to transform an alreadytransgenic Arabidopsis thaliana line which was homozygous for the T-DNAfrom the GA7 construct and whose seed contained approximately 15% DHA inseed lipids (Petrie et al., 2012). For this, use was made of thekanamycin selectable marker gene in the 35S:Moral-LPAAT1 construct whichwas different to the bar selectable marker gene already present in thetransgenic line. Transgenic seedlings were selected which were resistantto kanamycin and grown to maturity in a glasshouse. T2 seeds wereharvested and the fatty acid composition of their total seed lipidsanalysed by GC (Table 15). Three phenotypes were observed amongst the 33independently transformed lines. In a first group (6/33 lines), DPAincreased significantly to a level substantially greater than the levelof DHA, up to about 10.6% of total seed lipids. This came at the expenseof DHA which was strongly decreased in this group of lines. In two ofthe lines in this first group, the sum of DPA+DHA was reduced, but notin the other 4 lines. In a second group (5/33), the levels of DPA andDHA were about equal, with the sum of DPA+DHA about the same as for theparental seed. In the third group, the levels of DPA and DHA weresimilar to those in the parental seeds. One possible explanation for theincreased level of DPA in the first and second groups is that the LPAATout-competes the Δ4-desaturase for DPA-CoA substrate and preferentiallyincorporates the DPA into PA and thence into TAG, relative to theΔ4-desaturation. A second possible explanation is that theΔ4-desaturation is partially inhibited.

Seed from the Arabidopsis plants transformed with the T-DNA of the GA7construct which had been further transformed with the Cnl1::Moral-LPAATvector were harvested and oil extracted from the seed. The TAG fractionwas then isolated from the extracted oil by TLC methods and recoveredfrom the TLC plate. These TAG samples and samples of the seedoil priorto the fractionation were analysed by digestion with Rhizopus lipase todetermine the positional distribution of the DHA. The lipase is specificfor acyl groups esterified at the sn-1 or sn-3 position of TAG. This wasperformed by emulsifying each lipid sample in 5% gum arabic using anultrasonicator, adding the Rhizopus lipase solution in 0.1M Tris-HCl pH7.7 containing 5 mM CaCl₂ and incubating the mixtures at 30° C. withcontinuous shaking. Each reaction was stopped by adding chloroform:methanol (2/1, v/v) and one volume of 0.1M KCl to each mixture. Thelipid was extracted into the chloroform fraction and the relativeamounts determined of the sn-2 MAG, sn-1/3 FFA, DAG and TAG componentsof the resulting lipid by separation on 2.3% boric acid impregnated TLCusing hexane/diethylether/acetic acid (50/50/1, v/v). Lipid bands werevisualized by spraying 0.01% primuline in acetone/water (80/20, v/v)onto the TLC plate and visualisation under UV light. Individual lipidbands were identified on the basis of lipid standard spots, resolved onthe same TLC plate. TLC lipid bands were collected into glass vials andtheir fatty acid methyl esters were prepared using 1N methanolic-HCl(Supelco) and incubating at 80° C. for 2 h. Fatty acid composition ofindividual lipids were analysed by GC.

This assay demonstrated that the DHA in the parental seeds transformedwith the GA7 (lines 22-2-1-1 and 22-2-38-7) was preferentiallyesterified at the sn-1 or sn-3 position of the TAG. In contrast, the DHAin the NY11 and NY15 seed transformed with both the GA7 constructs andthe transgene encoding LPAAT was enriched at the sn-2 position, with 35%of the DHA in one of the lines and 48% of the DHA in the other linebeing esterified at the sn-2 position of TAG i.e. after lipase digestionthe DHA was present as sn-2-MAG (Table 16). Analogous results areobtained for B. napus and B. juncea seeds transformed with both theT-DNA from the GA7-modB construct and the LPAAT-encoding gene andproducing DHA.

In order to determine whether the Mortierella LPAAT or another LPAAT hadpreference for either DPA-CoA or DHA-CoA, in vitro reactions are set upby separately adding ¹⁴C-labelled-DPA-CoA or -DHA-CoA to lysates of N.benthamiana leaves transiently expressing the candidate LPAAT undercontrol of a constitutive promoter as described above. Reactions areincubated at 25° C. and the level of incorporation of the RC labelledfatty acids into PA determined by TLC analysis of the lipids. Theability of each LPAAT to use DHA-CoA relative to DPA-CoA is assessed.Genes encoding LPAATs which are confirmed to have good DHA incorporatingLPAAT activity are used to produced transformed DHA-producing canolaplants and seed.

Genes encoding LPAATs which have strong activity using DPA-CoA are usedto transform DPA-producing plants and seed, to increase the amount ofDPA esterified at the sn-2 position of TAG.

TABLE 15 Fatty acid composition (% of total fatty acids) of transgenicA. thaliana seeds transformed with an LPAAT1 construct as well as theGA7 construct for DHA production. C16:0 C18:0 C18:1 C18:1d11 C18:2C18:3n6 C18:3n3 C20:0 18:4n3 C20:1d11 NY-1 9.3 3.2 9.1 6.8 9.4 0.5 23.81.6 4.1 7.9 NY-2 10.7 3.3 6.5 4.4 7.6 0.3 28.1 1.9 4.3 8.5 NY-3 9.3 2.86.3 3.4 10.3 0.2 32.8 2.2 2.7 6.2 NY-4 11.4 3.5 4.5 3.1 7.0 0.3 32.5 2.14.7 5.5 NY-5 14.6 4.5 7.0 7.7 6.7 0.3 20.7 2.2 5.7 5.4 NY-6 7.8 2.7 12.52.2 18.0 0.1 24.9 1.8 0.7 15.5 NY-7 9.3 2.9 6.7 3.8 9.2 0.2 31.5 2.1 3.27.5 NY-8 8.8 3.2 8.2 5.5 11.0 0.3 25.3 1.9 3.0 8.3 NY-9 12.3 3.7 5.0 4.67.1 0.2 28.3 2.3 4.2 5.6 NY-10 8.6 3.2 8.5 3.1 9.7 0.3 31.5 1.6 3.4 8.7NY-11 11.5 3.2 4.5 2.5 7.1 0.3 33.3 2.1 3.9 5.7 NY-12 8.7 3.2 7.5 5.18.5 0.2 26.8 2.0 3.7 8.7 NY-13 11.5 3.4 5.2 3.4 8.3 0.3 30.0 2.2 5.0 6.2NY-14 9.2 2.9 6.6 2.0 10.3 0.2 34.7 1.9 3.3 7.7 NY-15 10.9 3.3 4.6 2.77.0 0.3 34.1 1.9 5.1 5.5 NY-16 10.5 3.4 6.0 4.6 7.8 0.3 30.3 1.8 4.4 5.4NY-17 9.1 2.4 5.9 2.5 10.4 0.2 35.4 1.6 3.6 6.4 NY-18 9.7 3.6 8.8 6.212.1 0.3 21.0 1.9 4.0 8.3 NY-19 8.4 3.1 12.0 3.1 14.6 0.2 28.8 1.7 1.611.3 NY-20 10.1 3.2 5.4 3.3 8.9 0.3 32.8 2.1 4.1 5.5 NY-21 10.5 3.6 5.63.8 8.2 0.3 31.9 2.0 4.6 5.9 NY-22 8.4 3.3 7.4 2.3 9.4 0.2 33.5 1.8 3.48.8 NY-23 8.3 2.8 7.0 1.9 11.0 0.2 34.6 1.9 2.6 9.3 NY-24 9.0 3.3 7.04.3 9.9 0.2 30.0 1.8 3.2 7.7 NY-25 9.4 3.3 6.0 3.6 8.2 0.2 32.6 1.8 4.06.8 NY-26 10.4 4.2 8.0 3.8 16.0 0.4 18.7 2.5 2.5 10.1 NY-27 9.4 3.2 7.55.3 11.4 0.2 28.6 2.0 2.3 7.5 NY-28 9.4 3.4 6.5 3.6 8.8 0.3 32.4 1.8 3.96.7 NY-29 10.2 3.7 7.6 4.3 8.0 0.4 28.8 1.7 4.8 7.6 NY-30 11.1 3.5 5.44.1 7.3 0.3 30.2 2.0 4.7 6.0 NY-31 9.6 3.0 5.6 2.1 8.5 0.2 35.4 2.0 3.97.1 NY-32 8.5 3.1 8.0 1.9 9.5 0.3 31.7 1.5 3.3 12.9 NY-33 10.3 3.8 7.76.3 8.1 0.3 24.4 2.0 4.4 7.5 20:1d13 C20:2n6 C20:4n6 C20:3n3 C22:020:4n3 C22:1 20:5n3 22:5n3 C22:6n3 NY-1 5.1 0.6 0.0 0.9 0.4 0.6 0.6 1.27.9 4.5 NY-2 3.7 0.7 0.0 1.1 0.5 1.1 0.8 1.4 1.1 11.6 NY-3 3.6 1.1 0.01.9 0.5 1.4 0.9 0.7 1.0 10.7 NY-4 2.3 1.0 0.0 1.9 0.6 0.8 0.6 1.1 0.914.3 NY-5 4.8 0.4 0.0 0.9 0.9 0.8 0.4 1.2 1.0 11.7 NY-6 3.1 1.4 0.0 1.20.3 0.5 1.5 0.3 3.0 0.8 NY-7 3.7 0.9 0.0 1.6 0.5 1.3 0.8 0.8 1.1 10.9NY-8 5.4 1.0 0.0 1.2 0.5 0.8 0.8 0.8 6.1 6.0 NY-9 3.8 0.8 0.0 1.6 0.70.7 0.6 1.1 1.2 13.8 NY-10 2.8 1.0 0.0 1.3 0.3 0.9 0.6 1.1 10.6 1.0NY-11 1.9 0.9 0.0 2.0 0.5 0.7 0.7 0.8 1.0 15.6 NY-12 5.1 0.9 0.0 1.2 0.51.1 0.8 1.2 10.0 2.6 NY-13 3.2 0.9 0.0 1.7 0.6 1.5 0.8 1.1 1.0 11.6NY-14 1.6 1.2 0.0 1.8 0.4 1.2 0.8 0.9 0.8 11.1 NY-15 2.0 0.9 0.0 1.8 0.50.8 0.5 1.0 1.0 14.7 NY-16 2.9 0.7 0.0 1.5 0.5 0.9 0.5 1.1 1.3 14.2NY-17 2.1 1.1 0.0 1.9 0.4 1.2 0.7 1.0 0.9 11.7 NY-18 5.9 0.8 0.0 0.9 0.50.6 0.9 1.0 5.7 5.1 NY-19 3.2 1.0 0.0 1.4 0.4 0.6 1.0 0.6 3.9 1.2 NY-202.8 1.0 0.0 1.9 0.5 1.1 0.7 0.9 1.1 12.1 NY-21 2.8 0.9 0.0 1.7 0.5 0.80.6 1.0 0.9 12.5 NY-22 2.2 1.2 0.0 1.7 0.4 1.3 0.7 1.0 5.5 6.1 NY-23 1.71.4 0.0 2.0 0.4 1.2 1.0 0.7 0.7 9.9 NY-24 4.3 1.0 0.0 1.6 0.4 0.6 0.80.8 3.4 8.8 NY-25 3.6 1.0 0.0 1.7 0.4 0.6 0.7 0.9 4.8 8.7 NY-26 4.0 1.00.0 0.8 0.8 1.9 1.0 1.4 1.4 8.4 NY-27 5.5 1.0 0.0 1.8 0.5 0.6 0.9 0.61.5 7.6 NY-28 3.3 0.9 0.0 1.6 0.4 0.7 0.6 1.0 10.1 2.7 NY-29 2.9 0.7 0.01.1 0.4 0.7 0.5 1.4 1.9 11.6 NY-30 3.0 0.8 0.0 1.7 0.5 0.7 0.7 1.1 1.013.7 NY-31 1.7 1.2 0.0 2.1 0.4 0.9 0.8 0.8 0.8 12.3 NY-32 1.4 1.0 0.11.1 0.3 1.2 0.8 1.2 0.8 9.8 NY-33 4.8 0.7 0.0 1.1 0.5 0.6 0.6 1.1 2.810.7

TABLE 16 Presence of DHA at the sn-2 position of TAG in the lipid fromtransgenic A. thaliana seeds transformed with Cnl1::Moral-LPAAT vectoras well as the T-DNA of the GA7 construct, relative to the presence ofDHA in TAG. The TAG and sn-2 MAG fatty acid compositions also contained0-0.4% each of 14:0, 16:1w13t, 16:2, 16:3, 22:0, and 24:0. Sample C16:016:1d9 C18:0 C18:1 C18:1d11 C18:2 C18:3n6 C:3n3 C20:0 18:4 C20:1d1120:1d13 22-2-1-1 12.2 0.4 4.4 6.4 3.9 7.2 0.8 28.8 1.6 4.3 9.7 2.3 TAG2-MAG 0.6 0.1 0.3 8.3 2.5 10.1 0.7 53.9 0.2 6.5 0.3 0.1 DHA at sn-2 =30% 22-2-38-7 10.0 0.2 3.7 6.0 2.7 6.4 0.4 33.8 1.6 3.7 11.3 1.8 oil2-MAG 0.5 0.1 0.3 9.7 2.4 11.1 0.6 60.0 0.1 3.6 0.3 0.1 DHA at sn-2 =19% Transformation additionally with gene encoding Mortierella alpinaLPAAT NY11- 11.0 0.2 3.4 6.0 2.8 9.2 0.3 34.8 1.6 3.6 6.3 1.8 TAG 2-MAG0.7 0.1 0.2 6.7 1.1 11.8 0.3 49.8 0.2 3.7 0.5 1.5 DHA at sn-2 = 48%NY-15-0 11.0 0.0 3.3 4.6 2.8 6.9 0.3 33.6 2.0 5.1 5.5 2.1 2-MAG 0.8 0.10.3 6.4 1.3 11.4 0.3 50.2 0.2 4.9 0.4 1.4 DHA at sn-2 = 37% SampleC20:2n6 C20:3n6 C20:4n6 C20:3n3 C20:4n3 C22:1 20:5n3 C22:4n6 22:5n622:4n3 22:5n3 C22:6n3 22-2-1-1 0.7 0.1 0.1 1.3 1.0 0.6 2.1 0.0 0.7 10.1TAG 2-MAG 0.1 0.0 0.0 0.3 0.2 0.0 3.8 0.0 2.3 9.1 DHA at sn-2 = 30%22-2-38-7 0.8 1.3 0.9 0.6 1.2 0.1 0.7 11.6 oil 2-MAG 0.1 0.0 0.0 0.4 0.20.0 2.1 0.1 1.3 6.7 DHA at sn-2 = 19% Transformation additionally withgene encoding Mortierella alpina LPAAT NY11- 1.0 0.0 0.0 1.8 0.7 0.6 0.90.0 0.1 0.6 12.2 TAG 2-MAG 0.3 0.0 0.0 1.6 0.6 0.1 0.8 0.1 0.2 1.6 17.8DHA at sn-2 = 48% NY-15-0 0.9 0.0 0.0 1.9 0.7 0.6 0.9 0.1 0.4 0.9 14.92-MAG 0.2 0.0 0.0 1.5 0.6 0.1 0.9 0.0 0.0 0.2 1.6 16.7 DHA at sn-2 = 37%

Example 8. Further Analysis of Transgenic Camelina sativa Seeds

Total Lipid Content

C. sativa seed which was homozygous for the T-DNA from the GA7 constructand containing DHA in its total fatty acid content was analysed for itstotal lipid content and composition as follows. Two consecutive solventextraction steps were performed on the seeds, firstly using hexane andsecondly using chloroform/methanol. No antioxidants were added duringthe extractions or analysis. The Soxhlet extraction method which iscommonly used to extract seed lipids by prolonged heating and refluxingof the lipid/solvent mixture was not used here because of the potentialfor degradation or oxidation of the ω3 PUFA such as DHA.

Hexane was used as the solvent in the first extraction since it is theindustry standard for oilseeds. Also, it preferentially extractsTAG-containing oil due to its solvating properties and its relativelypoor solubilization of polar lipids, particularly at room temperature.Transformed and control Camelina seeds (130 g and 30 g, respectively)were wetted with hexane and crushed using an electric agate mortar andpestle (Retsch Muhle, Germany). The mixtures were transferred toseparatory funnels and extracted four times using a total of 800 mLhexane, including an overnight static extraction for the thirdextraction. For each extraction, extracts were filtered to remove finesthrough a GFC glass fiber filter under vacuum, and then rotaryevaporated at 40° C. under vacuum. The extracts were pooled andconstituted the TAG-rich hexane extracts.

Following extraction with hexane, the remaining seed meals were furtherextracted using chloroform-methanol (CM, 1:1 v/v) using the procedure asfor the hexane extraction. The meal was then removed by filtration andthe combined extracts rotary evaporated. The pooled CM total crude lipidextracts were then dissolved using a one-phase methanol-chloroform-watermix (2:1:0.8 v/v/v). The phases were separated by the addition ofchloroform-water (final solvent ratio, 1:1:0.9 v/v/vmethanol-chloroform-water). The purified lipid in each extract waspartitioned in the lower chloroform phase, concentrated using rotaryevaporation and constituted the polar lipid-rich CM extracts. The lipidcontent in each of these extracts was determined gravimetrically.

For fatty acid compositional analysis, aliquots of the hexane and CMextracts were trans-methylated according to the method of Christie etal. (1982) to produce fatty acid methyl esters (FAME) usingmethanol-chloroform-conc. hydrochloric acid (3 mL, 10:1:1, 80° C., 2 h).FAME were extracted into hexane-chloroform (4:1, 3×1.8 mL). Samples ofthe remaining seed meal (1-2 g) after the hexane and CM extractions werealso trans-methylated to measure any residual lipid as FAME bygravimetry. The total lipid content of the seeds was calculated byadding the lipid contents of the hexane and CM extracts and the FAMEcontent of the transmethylated meal after solvent extraction.

The transgenic seeds contained slightly less total lipid at 36.2% ofseed weight compared to the wild-type Camelina sativa seeds at 40.9% ofseed weight. For seeds including oilseeds, the total lipid wasdetermined as the sum of solvent extractable lipid obtained byconsecutive extractions with hexane, then chloroform-methanol, plus theresidual lipid released by transmethylation of the extracted meal afterthe solvent extractions, as exemplified herein. This total lipidconsisted mainly of fatty acid containing lipids such astriacylglycerols and polar lipids and small amounts of non-fatty acidlipids e.g. phytosterols and fatty alcohols which may be present in thefree unesterified form or esterified with fatty acids. In addition, anysterol esters or wax esters and hydrocarbons such as carotenoids, forexample β-carotene, were also included in the solvent extractable lipidif present. These were included in the overall gravimetric determinationand were indicated in the TLC-FID analysis (Table 17).

Of the total lipid, 31%-38% of lipid per seed weight was extracted byhexane for the transgenic and control seeds, respectively, whichaccounted for 86% and 92% of the total lipid in the seeds. The CMextraction recovered a further 4.8% and 2.4% (of seed weight) mostlypolar lipid-rich extract from the transgenic and control seeds,respectively. The residual lipid released by transmethylation of theremaining solvent extracted oilseed meal was 0.3% and 0.4% of seedweight, respectively. That is, the first and second solvent extractionstogether extracted 99% of the total lipid content of the seeds (i.e. ofthe 36.2% or 40.9% of the seed weight, which was mostly fatty acidcontaining lipid such as triglycerides and polar lipids consisting ofglyco- and phospholipids (see next section-Lipid class analysis)).

Lipid Class Analysis

Lipid classes in the hexane and CM extracts were analyzed by thin-layerchromatography with flame-ionization detection (TLC-FID; Iatroscan MarkV, Iatron Laboratories, Tokyo, Japan) using hexane/diethyl ether/glacialacetic acid (70:10:0.1, v/v/v) as the developing solvent system incombination with Chromarod S-III silica on quartz rods. Suitablecalibration curves were prepared using representative standards obtainedfrom Nu-Chek Prep, Inc. (Elysian, Minn., USA). Data were processed usingSIC-48011 software (SISC Version: 7.0-E). Phospholipid species wereseparated by applying the purified phospholipid fraction obtained fromsilica column chromatography and developing the rods inchloroform/methanol/glacial acetic acid/water (85:17:5:2, v/v/v) priorto FID detection.

To separate TAG, glycolipid and phospholipid fractions from the CMextracts, silica gel 60 (100-200 mesh) (0.3-1 g) in a short glass columnor Pasteur pipette plugged with glass wool was used to purify 10 mg ofthe purified CM lipid extract. The residual TAG fraction in the CMextract was eluted using 20 mL of 10% diethyl ether in hexane, theglycolipids eluted with 20 mL of acetone and the phospholipids eluted intwo steps, first 10 mL of methanol then 10 mL ofmethanol-chloroform-water (5:3:2). This second elution increased therecovery of phospholipids. The yield of each fraction was determinedgravimetrically and the purity checked by TLC-FID. All extracts andfractions were stored in dichloromethane at −20° C. until furtheranalysis by GC and GC-MS.

The TAG-rich hexane extracts from each of the transgenic and controlseeds contained about 96% TAG. The CM extracts contained residual TAGamounting to 44% and 13% by weight of the CM extracts, respectively, forthe transgenic and wild-type seeds. In contrast to the hexane extracts,the CM extracts were rich in polar lipids, namely phospholipids andglycolipids, amounting to 50% and 76% by weight of the CM extracts,respectively, for the transgenic and control seeds (Table 17). The mainphospholipid was phosphatidyl choline (PC) and accounted for 70%-79% ofthe total phospholipids followed by phosphatidyl ethanolamine (PE,7%-13%) with relatively low levels of phosphatidic acid (PA, 2%-5%) andphosphatidyl serine (PS, <2%).

Fatty Acid Composition

Generally for seeds producing DHA and/or DPA, the inventors observedthat the fatty acid composition of the total lipids in the seeds asdetermined by direct transmethylation of all of the lipid in the seedwas similar to that of the TAG fraction. This was because more than 90%of the total lipids present in the seed occurred in the form of TAG.

The fatty acid composition of the different lipid classes in the hexaneand CM extracts was determined by gas chromatography (GC) and GC-MSanalysis using an Agilent Technologies 6890A GC instrument (Palo Alto,Calif., USA) fitted with a Supelco Equity™-1 fused silica capillarycolumn (15 m×0.1 mm i.d., 0.1 μm film thickness, Bellefont, Pa., USA),an FID, a split/splitless injector and an Agilent Technologies 7683BSeries auto sampler and injector. Helium was the carrier gas. Sampleswere injected in split-less mode at an oven temperature of 120° C. Afterinjection, the oven temperature was raised to 270° C. at 10° C. min⁻¹and finally to 300° C. at 5° C. min⁻¹. Eluted compounds were quantifiedwith Agilent Technologies ChemStation software (Palo Alto, Calif., USA).GC results were subject to an error of not more than ±5% of individualcomponent areas.

TABLE 17 Lipid class composition (% of total lipid obtained for eachextraction step) of hexane and CM extracts from transgenic and controlCamelina sativa seeds. SE, WE and HC were not separated from each other.Transgenic seeds Control seeds Lipid class Hexane CM Hexane CM SE/WE/HC*1.0 1.4 1.0 1.4 TAG 95.6 44.2 96.0 13.1 FFA 0.9 1.3 0.8 1.4 UN** 0.9 1.10.8 1.2 ST 0.5 0.7 0.4 0.4 MAG 0.7 1.1 0.8 6.2 PL 0.3 50.3 0.3 76.3Total 100.0 100.0 100.0 100.0 Abbreviations: sterol esters (SE), waxesters (WE). hydrocarbons (HC), triacylglycerols (TAG), free fatty acids(FFA), unknown (UN), sterols (ST), monoacylglycerols (MAG), polar lipids(PL) consisting of glycolipids and phospholipids;  *SE, WE and HCco-elute with this system; **May contain fatty alcohols anddiacylglycerols (DAG).

GC-mass spectrometric (GC-MS) analyses were performed on a FinniganTrace ultra Quadrupole GC-MS (model: ThermoQuest Trace DSQ, ThermoElectron Corporation). Data were processed with ThermoQuest Xcalibursoftware (Austin, Tex., USA). The GC was fitted with an on-columninjector and a capillary HP-5 Ultra Agilient J & W column (50 m×0.32 mmi.d., 0.17 μm film thickness, Agilent Technologies, Santa Clara, Calif.,USA) of similar polarity to that described above. Individual componentswere identified using mass spectral data and by comparing retention timedata with those obtained for authentic and laboratory standards. A fullprocedural blank analysis was performed concurrent to the sample batch.

The data for the fatty acid composition in the different lipid classesin the extracts are shown in Table 18. In the DHA-producing Camelinaseed, the DHA was distributed in the major lipid fractions (TAG,phospholipids and glycolipids) at a proportion ranging between 1.6% and6.8% with an inverse relationship between the proportions of DHA andALA. The TAG-rich hexane extract from the transgenic seed contained 6.8%DHA and 41% ALA (Table 18). The polar lipid-rich CM extract contained4.2% DHA and 50% ALA i.e. relatively less DHA and more ALA. Residual TAGfrom the polar lipid-rich CM extract contained 6% DHA and 40% ALA. Theglycolipid fraction isolated from the CM extract contained 3% DHA and39% ALA and the phospholipid fraction contained the lowest level of DHA(1.6%) and the highest levels of ALA (54%). The transgenic Camelina seedcontained higher levels of ALA and lower levels of LA (linoleic acid,18:2ω6) compared with the control seeds in the major lipid classes (TAG,glycolipids and phospholipids). The proportions of ALA and LA were: ALA39%-54% and LA 4%-9% for transgenic seeds and ALA 12%-32% and LA 20%-29%for control seeds. The relative level of erucic acid (22:1ω9) was lowerin all fractions in the transgenic seeds than in the control seeds, forexample, in the hexane extracts 1.3% versus 2.7% (Table 18).

Sterol Composition in the Seeds

To determine the sterol content and composition in the extracted lipids,samples of approximately 10 mg total lipid from the TAG-rich hexaneextract and the polar lipid-rich CM extract were saponified using 4 mL5% KOH in 80% MeOH and heated for 2 h at 80° C. in a Teflon-linedscrew-capped glass test tube. After the reaction mixtures were cooled, 2mL of Milli-Q water was added and the sterols and alcohols wereextracted three times into 2 mL of hexane:dichloromethane (4:1, v/v) byshaking and vortexing. The mixtures were centrifuged and each extract inthe organic phase was washed with 2 mL of Milli-Q water by shaking andcentrifugation. After taking off the top sterol-containing organiclayer, the solvent was evaporated using a stream of nitrogen gas and thesterols and alcohols silylated using 200 μl, ofBis(trimethylsilyl)-trifluoroacetamide (BSTFA, Sigma-Aldrich) by heatingfor 2 h at 80° C. in a sealed GC vial. By this method, free hydroxylgroups were converted to their trimethylsilyl ethers. The sterol- andalcohol-OTMSi derivatives were dried under a stream of nitrogen gas on aheating block (40° C.) and re-dissolved in dichloromethane (DCM)immediately prior to GC/GC-MS analysis as described above.

TABLE 18 Fatty acid composition (% of total fatty acids) of lipidextracts and fractions of transgenic and control C. sativa seeds.Transgenic seeds Control seeds Hexane CM Meal Hexane CM Meal Fatty acidTAG Total TAG GL PL Residue TAG Total TAG GL PL Residue 16:1ω7 0.1 0.20.1 0.2 0.1 0.2 0.1 0.2 0.2 — — 0.3 16:0 6.2 12.8 6.8 21.3 19.4 10.4 6.712.8 7.8 29.6 13.7 10.3 18:4ω3 3.7 3.3 3.4 2.1 2.9 3.6 — — — — — —18:2ω6 7.1 3.9 8.8 7.2 3.7 8.8 22.2 28.4 29.4 20.8 29.3 27.9 18:3ω3 41.950.3 39.9 38.6 54.1 38.9 32.0 20.6 19.7 13.0 12.3 20.0 18:1ω9 11.1 4.79.6 7.2 2.8 8.1 14.0 25.4 13.3 14.7 35.7 14.3 18:1ω7 1.4 2.3 2.1 3.7 3.42.8 1.0 1.5 2.2 4.0 2.8 2.2 18:0 3.2 4.0 3.0 4.5 5.7 3.1 3.0 2.7 2.9 5.73.6 2.7 20:5ω3 0.4 0.2 0.3 — — 0.3 — — — — — — 20:4ω3 0.4 0.4 0.4 — 0.20.3 — — — — — — 20:2ω6 0.7 0.7 0.8 0.6 0.4 0.7 1.8 0.8 2.1 1.2 — 1.820:3ω3 0.8 1.2 0.9 0.6 1.3 0.5 0.9 0.3 — — — 0.4 20:1ω9/11 11.6 6.1 10.95.1 1.3 8.4 12.5 3.0 11.1 4.2 1.7 9.4 20:1ω7 0.6 0.8 1.4 0.6 0.2 1.1 0.60.6 2.0 1.3 — 1.8 20:0 1.3 0.8 1.4 0.6 0.1 1.4 1.5 0.7 2.0 1.4 — 1.822:6ω 6.8 4.2 6.1 3.0 1.6 5.4 — — — — — — 22:5ω3 0.3 1.1 0.4 0.6 1.4 0.3— — — — — — 22:1ω9 1.3 1.0 1.8 0.6 0.1 1.5 2.7 0.7 3.6 0.9 — 2.9 22:00.3 0.2 0.3 0.6 0.1 0.7 0.3 0.2 0.7 0.8 — 0.8 24:1ω9 0.3 0.4 0.4 0.6 0.30.6 0.3 0.6 0.7 0.9 0.5 1.0 24:0 0.1 0.4 0.2 0.9 0.4 1.1 0.1 0.4 0.5 1.40.4 1.3 others * 0.4 1.0 1.0 1.4 0.5 1.8 0.3 1.1 0.9 0.1 — 1.1 Total 100100 100 100 100 100 100 100 100 100 100 100 Abbreviations:triacylglycerols (TAG), glycolipids (GL), phospholipids (PL); Total:polar lipid-rich extract containing GL and PL from CM extraction; TAG,GL and PL were separated by silica column chromatography of the CMextracts; * Sum of minor fatty acids

The major sterols in both the transgenic and control seeds were24-ethylcholesterol (sitosterol, 43%-54% of the total sterols),24-methyicholesterol (campesterol, 20%-26%) with lower levels ofcholesterol (5%-8%), brassicasterol (2%-7%), isofucosterol(Δ5-avenasterol, 4%-6%), stigmasterol (0.5%-3%), cholest-7-en-3β-ol,(0.2%-0.5%), 24-methylcholestanol (campestanol, 0.4%-1%) and24-dehydrocholesterol (0.5%-2%) (Table 19). These nine sterols accountedfor 86%-95% of the total sterols, with the remaining components beingsterols only partially identified for the numbers of carbons and doublebonds. The overall sterol profiles were similar between the transgenicand control seeds for both the hexane and CM extracts.

Fatty Alcohol Analysis

Fatty alcohols in the extracts were derivatised and analysed as for thesterols. A series of fatty alcohols from C₁₆-C₂₂, with accompanyingiso-branched fatty alcohols, were identified in both the hexane and CMextracts. Similar profiles were observed for the transgenic and controlseeds, with some variation in the proportions of individual componentsobserved. Phytol, derived from chlorophyll, was the major aliphaticalcohol and accounted for 47% and 37% of the total fatty alcohols in thehexane fractions in the transgenic and control seeds, respectively. Theodd-chain alcohols were present at higher levels in the CM extract(37%-38% of the total fatty alcohol content) than in the hexane extract(16%-23%). Iso-17:0 (16%-38%) predominated over 17:0 (0.3%-5.7%).Another odd-chain alcohol present was 19:0 (4.5%-6.5%). Other alcoholsdetected included iso-16:0, 16:0, iso-18:0, 18:1, 18:0, with minorlevels of iso-20:0, 20:1, 20:0, iso-22:0, 22:1 and 22:0 also present.

Discussion

The results indicated that crushing using a motorized mortar and pestlewith multiple extractions with hexane at room temperature was effectivein recovering most of the TAG-containing oil from the transgenic seeds.In addition to the oil from the transgenic seeds containing moderatelevels of DHA, the transgenic seeds also had markedly higher levels ofALA in the major lipid classes (triacylglycerols, glycolipids andphospholipids) compared with the control seeds. This showed that theΔ15-desaturase activity was considerably enhanced in the transgenicseeds during seed development. Interestingly, there were some slightdifferences in the fatty acid composition and proportion of DHA in thevarious extracts and fractions with the DHA levels being higher in theTAG-rich hexane extract and TAG from CM extraction (6%-6.8%) and lowerin the polar lipid fractions (3% in glycolipids and 1.6% inphospholipids). The level of 16:0 was higher in the polar lipidfractions of glycolipids and phospholipids in the CM extracts (19%-21%)compared with the TAG-rich hexane extract and TAG from CM extraction(6%-7%).

TABLE 19 Sterol composition (% of total sterols) of transgenic andcontrol Camelina seeds. Transgenic seeds Control seeds Sterols Hexane CMHexane CM 24-dehydrocholesterol 0.8 1.8 0.5 1.4 cholesterol 5.7 7.6 4.77.2 brassicasterol 4.4 6.5 1.9 4.2 cholest-7-en-3β-ol 0.2 0.5 0.3 0.4campesterol 24.5 20.8 25.7 21.7 campestanol 0.4 1.1 0.4 0.9 stigmasterol1.0 2.6 0.5 1.6 sitosterol 54.3 43.7 53.8 42.9Δ5-avenasterol(isofucosterol) 4.2 5.2 4.7 5.5 Sum 95.5 89.6 92.6 85.9Others UN1 C28 1 db 0.6 1.2 0.7 1.2 UN2 C29 1 db 1.2 2.0 1.2 2.4 UN3 C292 db 0.9 1.8 1.3 2.4 UN4 C28 1 db 0.3 0.9 0.6 1.1 UN5 C30 2 db 1.2 1.81.4 1.8 UN6 C29 1 db + C30 2db 0.3 2.7 2.2 5.2 Sum of others 4.5 10.47.4 14.1 Total 100 100 100 100 Abbreviations: UN denotes unknown sterol,the number after C indicates the number of carbon atoms and db denotesnumber of double bonds

The sterol composition of the transgenic seeds and control seeds weresimilar to that found in refined Camelina oil (Shukla et al., 2002) withthe same major sterols present, indicating that the added genes did notaffect sterol synthesis in the seeds. The level of cholesterol inCamelina oil was higher than occurred in most vegetable oils. Brassicasterol was present, which is a characteristic sterol found in theBrassicaceae family which includes Camelina sativa.

Example 9. Production of LC-PUFA in Brassica juncea Seeds

Transgenic Brassica juncea plants were produced using the GA7-modBconstruct (Example 4) for the production of DHA, as follows. B. junceaseeds of a long-daylength sensitive variety were sterilized usingchlorine gas as described by Kereszt et al. (2007). Sterilized seedswere germinated on ½ strength MS media (Murashige and Skoog, 1962)solidified with 0.8% agar, adjusted to pH 5.8 and grown at 24° C. underfluorescent lighting (50 μE/m² s) with a 16/8 hour (light/dark)photoperiod for 6-7 days. Cotyledonary petioles with 2-4 mm stalk wereisolated aseptically from these seedlings and used as explants.Agrobacterium tumefaciens strain AGL1 was transformed with the binaryconstruct GA7. Agrobacterium culture was initiated and processed forinfection as described by Betide et al. (2013). For all transformations,about 50 freshly-isolated cotyledonary petioles were infected with 10 mlof A. tumefaciens culture for 6 minutes. The infected petioles wereblotted on sterile filter paper to remove excess A. tumefaciens andtransferred to co-cultivation media (MS containing 1.5 mg/L BA, 0.01mg/L NAA and 100 pM acetosyringone, also supplemented with L-cysteine(50 mg/L), ascorbic acid (15 mg/L) and MES (250 mg/L). All plates weresealed with micropore tape and incubated in the dark at 24° C. for 48hours of co-cultivation. The explants were then transferred topre-selection medium (MS-agar containing 1.5 mg/L BA, 0.01 mg/L NAA, 3mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for4-5 days at 24° C. with a 16/8 hour photoperiod before the explants weretransferred to selection medium (MS-agar containing 1.5 mg/L BA, 0.01mg/L NAA, 3 mg/L AgNO₃, 250 mg/L cefotaxime, 50 mg/L timentin and 5 mg/LPPT) and cultured for 4 weeks at 24° C. with 16/8 hour photoperiod.Explants with green callus were transferred to shoot regeneration medium(MS-agar containing 2.0 mg/L BA, 3 mg/L AgNO₃, 250 mg/L cefotaxime, 50mg/L timentin and 5 mg/L PPT) and cultured for another 2 weeks. Smallregenerating shoot buds were transferred to hormone free MS medium(MS-agar containing 3 mg/L AgNO₃, 250 mg/L cefotaxime, 50 mg/L timentinand 5 mg/L PPT) and cultured for another 2-3 weeks.

Potential transgenic shoots of at least 1.5 cm in size were isolated andtransferred to root induction medium (MS-agar containing 0.5 mg/L NAA, 3mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for2-3 weeks. Transgenic shoots confirmed by PCR and having prolific rootswere transferred to soil in a greenhouse and grown under a photoperiodof 16/8 h (light/dark) at 22° C. Three confirmed transgenic plants wereobtained. The transformed plants were grown in the greenhouse, allowedto self-fertilise, and T1 seed harvested. The fatty acid composition wasanalysed of the lipid from pools of T1 seeds from each TO transformedplants, which showed the presence of 2.8% DPA and 7.2% DHA in one linedesignated JT1-4, whereas another line designated JT1-6 exhibited 2.6%DPA.

Seedoil from individual T1 seeds was analysed for fatty acidcomposition; some of the data is shown in Table 20. Several T1 seedsproduced DHA at a level of 10% to about 21% of the total fatty acidcontent, including JT1-4-A-13, JT1-4-A -5, and JT1-4-B-13. Surprisinglyand unexpectedly, some of the T1 seeds contained DPA at levels of 10% toabout 18% of the total fatty acid content and no detectable DHA (<0.1%).One possible explanation for these seeds is that the Δ4-desaturase genein the T-DNA inserted in these plants was inactivated, perhaps through aspontaneous mutation. T1 seeds were germinated and one emerged cotyledonfrom each analysed for fatty acid composition in the remaining oil. Theremainder of each seedling was maintained and grown to maturity toprovide T2 seed.

Transgenic plants which were homozygous for single T-DNA insertions wereidentified and selected. Plants of one selected line designated JT1-4-17had a single T-DNA insertion and produced DHA with only low levels ofDPA, whereas those of a second selected line designated JT1-4-34 alsohad a single T-DNA insertion but produced DPA without producing DHA. Theinventors concluded that the original transformant contained twoseparate T-DNAs, one which conferred production of DHA and the otherwhich conferred production of DPA without DHA. The B. juncea plantsproducing DHA in their seeds were crossed with the plants producing DPAin their seeds. The F1 progeny included plants which were heterozygousfor both of the T-DNA insertions. Seed from these progeny plants wereobserved to produce about 20% DHA and about 6% DPA, for a total DHA+DPAcontent of 26%. The F1 plants are self-fertilised and progeny which arehomozygous for both of the T-DNA insertions are expected to produce upto 35% DHA and DPA.

About 18% DPA was observed in the lipid of pooled seed of the T3 progenydesignated JT1-4-34-11. Similarly about 17.5% DHA was observed in thelipid from pooled seed in the progeny of T3 JT1-4-17-20. Fatty acidcompositions of JT1-4 T1 pooled seed, T1 single seed, T2 pooled seed, T2single seed, and T3 pooled seed, T3 single seed are in Tables 21 to 24.JT1-4 T3 segregant JT-1-4-34-11, had a pooled T3 seed DPA content of 18%and the single seed from this particular segregant had a DPA content ofabout 26%, each as a percentage of the total fatty acid content.

The following parameters were calculated for oil from a seed having17.9% DPA: total saturated fatty acids, 6.8%; total monounsaturatedfatty acids, 36.7%; total polyunsaturated fatty acids, 56.6%, total ω6fatty acids, 7.1%; new ω6 fatty acids, 0.4% of which all was GLA; totalω3 fatty acids, 46.5%; new ω3 fatty acids, 24.0%; ratio of total ω6:total ω3 fatty acids, 6.5; ratio of new ω6: new ω3 fatty acids, 60; theefficiency of conversion of oleic acid to LA by Δ12-desaturase, 61%; theefficiency of conversion of ALA to SDA by Δ6-desaturase, 51%; theefficiency of conversion of SDA to ETA acid by Δ6-elongase, 90%; theefficiency of conversion of ETA to EPA by Δ5-desaturase, 87%; theefficiency of conversion of EPA to DPA by Δ5-elongase, 98%.

In order to produce more transgenic plants in B. juncea with the modBconstruct, the transformation was repeated five times and 35 presumedtransgenic shoots/seedlings were regenerated. T1 seed analysis iscarried out to determine DPA and DHA content.

In order to produce further seed containing DPA and no DHA, theΔ4-desaturase gene was deleted from the modB construct and the resultantconstruct used to transform B. juncea and B. napus. Progeny seed with upto 35% DPA in the total fatty acid content of the seed lipid areproduced.

When the oil extracted from the seeds of a plant producing DHA wasexamined by NMR, at least 95% of the DHA was observed to be present atthe sn-1,3 position of the TAG molecules.

TABLE 20 Fatty acid composition of seedoil from T1 seeds of B. junceatransformed with the 1-DNA from GA7. T1 seed No. C16:0 16:1d9 C18:0C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3 C20:0 JT1-4-A-1 5.0 0.2 2.7 23.53.4 17.0 0.7 24.8 0.7 JT1-4-A-2 4.3 0.3 2.6 37.2 3.2 11.0 0.3 22.1 0.7JT1-4-A-3 5.6 0.3 2.7 20.8 3.7 16.0 0.6 24.4 0.7 JT1-4-A-4 4.6 0.4 2.836.2 3.4 10.6 0.3 24.5 0.8 JT1-4-A-5 5.0 0.2 3.2 20.3 3.6 13.7 0.7 25.90.7 JT1-4-A-6 4.8 0.4 3.4 37.9 3.7 7.4 0.4 19.9 0.9 JT1-4-A-7 5.6 0.33.0 26.2 4.0 8.9 0.3 26.6 0.6 JT1-4-A-8 4.8 0.4 2.9 40.3 3.4 7.8 0.322.2 0.8 JT1-4-A-9 7.1 0.3 3.6 17.7 4.3 17.9 0.7 23.1 1.0 JT1-4-A-10 5.10.2 4.2 22.3 3.4 19.5 0.7 21.7 0.8 JT1-4-A-11 5.0 0.5 2.8 37.6 4.0 7.10.4 19.2 0.7 JT1-4-A-12 5.2 0.3 3.0 28.2 4.0 9.2 0.3 27.4 0.6 JT1-4-A-135.4 0.2 3.0 16.7 4.1 9.9 0.6 29.9 0.7 JT1-4-A-14 5.1 0.4 3.1 30.0 4.011.5 0.3 27.7 0.7 JT1-4-A-15 5.1 0.4 2.5 34.2 3.6 6.9 0.6 20.4 0.7JT1-4-B-1 5.5 0.2 2.7 18.9 4.0 17.6 0.8 24.1 0.8 JT1-4-B-2 5.5 0.2 2.720.2 4.0 14.3 0.5 25.5 0.7 JT1-4-B-3 5.3 0.3 3.6 34.1 3.5 35.0 0.6 9.30.8 JT1-4-B-4 5.3 0.3 3.1 25.2 3.6 17.0 0.7 24.1 0.7 JT1-4-B-5 5.5 0.52.2 30.1 4.6 10.2 0.5 21.7 0.6 JT1-4-B-6 5.6 0.3 2.5 19.5 3.8 15.2 0.527.7 0.6 JT1-4-B-7 5.9 0.5 2.0 29.9 4.0 11.2 0.3 26.2 0.6 JT1-4-B-8 6.20.5 1-.9 33.1 4.0 30.0 0.5 12.7 0.6 JT1-4-B-9 4.9 0.2 3.4 24.6 3.0 18.50.3 26.2 0.8 JT1-4-B-10 5.2 0.3 2.7 19.0 4.0 12.0 0.6 30.5 0.7JT1-4-6-11 4.8 0.2 3.0 23.7 3.1 18.1 0.6 23.5 0.7 JT1-4-B-12 5.0 0.2 2.619.6 3.4 12.5 0.6 26.9 0.8 JT1-4-B-13 5.6 0.3 2.8 20.9 3.9 11.9 0.4 27.00.7 JT1-4-B-14 5.1 0.3 3.1 25.5 3.3 16.7 0.7 23.9 0.8 JT1-4-B-15 5.6 0.32.7 19.5 4.1 14.0 0.8 24.6 0.7 T1 seed No. 18:4n3 C20:1d11 C20:2n6C20:3n3 20:4n3 20:5n3 22:5n3 C22:6n3 JT1-4-A-1 2.0 1.1 0.2 0.8 4.0 0.62.4 9.9 JT1-4-A-2 0.9 1.3 0.2 1.4 3.2 0.3 9.4 0.0 JT1-4-A-3 2.0 0.9 0.21.1 4.5 0.7 3.1 11.4 JT1-4-A-4 9.9 1.7 0.2 0.3 0.5 0.0 2.5 0.0 JT1-4-A-52.0 0.9 0.2 1.3 4.4 1.5 1.6 13.5 JT1-4-A-6 1.4 1.4 0.1 0.8 1.9 0.4 13.90.0 JT1-4-A-7 1.8 1.0 0.1 1.8 3.7 1.3 2.2 11.3 JT1-4-A-8 1.4 1.3 0.1 0.82.4 0.4 9.6 0.0 JT1-4-A-9 2.1 0.8 0.2 1.5 3.6 0.8 2.0 11.9 JT1-4-A-101.5 0.9 0.2 1.7 7.8 0.9 1.0 6.5 JT1-4-A-11 1.9 1.4 0.2 0.5 1.6 0.3 15.50.0 JT1-4-A-12 1.9 0.9 0.1 1.5 3.2 1.1 1.8 10.2 JT1-4-A-13 2.2 1.0 0.21.7 2.0 1.1 2.0 17.9 JT1-4-A-14 2.2 1.0 0.1 0.6 2.4 0.8 1.3 7.8JT1-4-A-15 1.6 1.1 0.2 0.6 4.7 0.9 15.2 0.0 JT1-4-B-1 2.2 1.0 0.2 1.24.6 0.9 2.2 11.5 JT1-4-B-2 1.7 0.9 0.2 1.6 8.7 1.3 2.2 8.5 JT1-4-B-3 0.21.4 0.4 0.6 0.9 0.1 0.3 2.1 JT1-4-B-4 1.9 1.0 0.2 0.8 4.3 0.5 2.3 7.8JT1-4-B-5 1.4 1.1 0.2 0.9 2.4 0.5 16.1 0.0 JT1-4-B-6 2.1 0.9 0.2 1.1 3.70.6 3.3 11.1 JT1-4-B-7 11.5 1.4 0.2 0.3 0.4 0.0 4.1 0.1 JT1-4-B-8 0.31.3 0.4 1.4 0.9 0.1 4.4 0.0 JT1-4-B-9 1.3 1.1 0.2 2.0 5.5 0.6 0.8 5.2JT1-4-B-10 1.6 1.0 0.2 1.7 4.9 1.1 3.0 10.2 JT1-4-6-11 1.6 1.2 0.2 1.54.5 0.8 1.6 9.6 JT1-4-B-12 3.1 1.1 0.2 0.9 5.6 0.9 3.5 11.7 JT1-4-B-132.0 1.0 0.2 1.7 2.3 0.7 4.1 13.5 JT1-4-B-14 1.8 1.2 0.2 0.9 2.6 0.4 2.99.2 JT1-4-B-15 2.7 0.9 0.2 0.7 9.4 1.3 2.5 8.5 The seedoil samples alsocontained 0.1% C14:0; 0.1-0.2% C16:3; 0.0-0.1% of each of C20:1Δ13,C20:3ω6 and C20:4ω6; 0.3-0.4% C22:0; no C22:1 and C22:2ω6; 0.2% C24:0and 0.2-0.4% C24:1.

TABLE 21 Fatty acid composition of lipid from T1 seeds (pooled) of B.juncea transformed with the T-DNA from GA7-modB. The lipids alsocontained about 0.1% of each of 14:0, 16:3, 20:1d13, and 16:2, 22:1 werenot detected. C16:0 C16:1 C18:0 C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3C20:0 18:4n3 C20:1d11 C20:2n6 JT1-2 4.2 0.3 2.5 42.4 3.2 27.7 0.1 16.40.6 0.0 1.2 0.1 JT1-3 4.5 0.3 2.7 44.6 3.1 26.8 0.1 14.8 0.7 0.0 1.2 0.1JT1-4 5.1 0.3 3.2 26.8 3.5 17.4 0.5 22.8 0.7 2.5 1.1 0.2 JT1-5 4.7 0.42.4 41.6 3.4 28.4 0.1 15.8 0.7 0.0 1.2 0.1 JT1-6 4.8 0.4 2.3 37.3 3.330.2 0.4 13.2 0.7 0.2 1.4 0.3 C20:3n6 C20:4n6 C20:3n3 C22:0 20:4n320:5n3 C22:2n6 22:3n3 C24:0 C24:1 22:5n3 C22:6n3 JT1-2 0.0 0.0 0.0 0.30.0 0.0 0.0 0.0 0.2 0.4 0.0 0.0 JT1-3 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.00.2 0.4 0.0 0.0 JT1-4 0.0 0.0 1.2 0.3 2.9 0.7 0.0 0.1 0.2 0.3 2.8 7.2JT1-5 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.2 0.4 0.0 0.0 JT1-6 0.0 0.0 0.70.3 0.6 0.1 0.0 0.3 0.2 0.5 2.6 0.0

TABLE 22 Fatty acid composition of seed oil from T1 (single) seeds of B.juncea transformed with the T-DNA from GA7-modB. T1 seed No. C16:016:1d9 C18:0 C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3 C20:0 JT1-4-A-1 5.00.2 2.7 23.5 3.4 17.0 0.7 24.8 0.7 JT1-4-A-2 4.3 0.3 2.6 37.2 3.2 11.00.3 22.1 0.7 JT1-4-A-3 5.6 0.3 2.7 20.8 3.7 16.0 0.6 24.4 0.7 JT1-4-A-44.6 0.4 2.8 36.2 3.4 10.6 0.3 24.5 0.8 JT1-4-A-5 5.0 0.2 3.2 20.3 3.613.7 0.7 25.9 0.7 JT1-4-A-6 4.8 0.4 3.4 37.9 3.7 7.4 0.4 19.9 0.9JT1-4-A-7 5.6 0.3 3.0 26.2 4.0 8.9 0.3 26.6 0.6 JT1-4-A-8 4.8 0.4 2.940.3 3.4 7.8 0.3 22.2 0.8 JT1-4-A-9 7.1 0.3 3.6 17.7 4.3 17.9 0.7 23.11.0 JT1-4-A-10 5.1 0.2 4.2 22.3 3.4 19.5 0.7 21.7 0.8 JT1-4-A-11 5.0 0.52.8 37.6 4.0 7.1 0.4 19.2 0.7 JT1-4-A-12 5.2 0.3 3.0 28.2 4.0 9.2 0.327.4 0.6 JT1-4-A-13 5.4 0.2 3.0 16.7 4.1 9.9 0.6 29.9 0.7 JT1-4-A-14 5.10.4 3.1 30.0 4.0 11.5 0.3 27.7 0.7 JT1-4-A-15 5.1 0.4 2.5 34.2 3.6 6.90.6 20.4 0.7 IT1-4-B-1 5.5 0.2 2.7 18.9 4.0 17.6 0.8 24.1 0.8 JT1-4-B-25.5 0.2 2.7 20.2 4.0 14.3 0.5 25.5 0.7 JT1-4-B-3 5.3 0.3 3.6 34.1 3.535.0 0.6 9.3 0.8 JT1-4-B-4 5.3 0.3 3.1 25.2 3.6 17.0 0.7 24.1 0.7JT1-4-B-5 5.5 0.5 2.2 30.1 4.6 10.2 0.5 21.7 0.6 JT1-4-B-6 5.6 0.3 2.519.5 3.8 15.2 0.5 27.7 0.6 JT1-4-B-7 5.9 0.5 2.0 29.9 4.0 11.2 0.3 26.20.6 JT1-4-B-8 6.2 0.5 1.9 33.1 4.0 30.0 0.5 12.7 0.6 JT1-4-B-9 4.9 0.23.4 24.6 3.0 18.5 0.3 26.2 0.8 JT1-4-B-10 5.2 0.3 2.7 19.0 4.0 12.0 0.630.5 0.7 JT1-4-B-11 4.8 0.2 3.0 23.7 3.1 18.1 0.6 23.5 0.7 JT1-4-B-125.0 0.2 2.6 19.6 3.4 12.5 0.6 26.9 0.8 JT1-4-B-13 5.6 0.3 2.8 20.9 3.911.9 0.4 27.0 0.7 JT1-4-B-14 5.1 0.3 3.1 25.5 3.3 16.7 0.7 23.9 0.8JT1-4-B-15 5.6 0.3 2.7 19.5 4.1 14.0 0.8 24.6 0.7 T1 seed No. 18:4n3C20:1d11 C20:2n6 C20:3n3 20:4n3 20:5n3 22:5n3 C22:6n3 JT1-4-A-1 2.0 1.10.2 0.8 4.0 0.6 2.4 9.9 JT1-4-A-2 0.9 1.3 0.2 1.4 3.2 0.3 9.4 0.0JT1-4-A-3 2.0 0.9 0.2 1.1 4.5 0.7 3.1 11.4 JT1-4-A-4 9.9 1.7 0.2 0.3 0.50.0 2.5 0.0 JT1-4-A-5 2.0 0.9 0.2 1.3 4.4 1.5 1.6 13.5 JT1-4-A-6 1.4 1.40.1 0.8 1.9 0.4 13.9 0.0 JT1-4-A-7 1.8 1.0 0.1 1.8 3.7 1.3 2.2 11.3JT1-4-A-8 1.4 1.3 0.1 0.8 2.4 0.4 9.6 0.0 JT1-4-A-9 2.1 0.8 0.2 1.5 3.60.8 2.0 11.9 JT1-4-A-10 1.5 0.9 0.2 1.7 7.8 0.9 1.0 6.5 JT1-4-A-11 1.91.4 0.2 0.5 1.6 0.3 15.5 0.0 JT1-4-A-12 1.9 0.9 0.1 1.5 3.2 1.1 1.8 10.2JT1-4-A-13 2.2 1.0 0.2 1.7 2.0 1.1 2.0 17.9 JT1-4-A-14 2.2 1.0 0.1 0.62.4 0.8 1.3 7.8 JT1-4-A-15 1.6 1.1 0.2 0.6 4.7 0.9 15.2 0.0 IT1-4-B-12.2 1.0 0.2 1.2 4.6 0.9 2.2 11.5 JT1-4-B-2 1.7 0.9 0.2 1.6 8.7 1.3 2.28.5 JT1-4-B-3 0.2 1.4 0.4 0.6 0.9 0.1 0.3 2.1 JT1-4-B-4 1.9 1.0 0.2 0.84.3 0.5 2.3 7.8 JT1-4-B-5 1.4 1.1 0.2 0.9 2.4 0.5 16.1 0.0 JT1-4-B-6 2.10.9 0.2 1.1 3.7 0.6 3.3 11.1 JT1-4-B-7 11.5 1.4 0.2 0.3 0.4 0.0 4.1 0.1JT1-4-B-8 0.3 1.3 0.4 1.4 0.9 0.1 4.4 0.0 JT1-4-B-9 1.3 1.1 0.2 2.0 5.50.6 0.8 5.2 JT1-4-B-10 1.6 1.0 0.2 1.7 4.9 1.1 3.0 10.2 JT1-4-B-11 1.61.2 0.2 1.5 4.5 0.8 1.6 9.6 JT1-4-B-12 3.1 1.1 0.2 0.9 5.6 0.9 3.5 11.7JT1-4-B-13 2.0 1.0 0.2 1.7 2.3 0.7 4.1 13.5 JT1-4-B-14 1.8 1.2 0.2 0.92.6 0.4 2.9 9.2 JT1-4-B-15 2.7 0.9 0.2 0.7 9.4 1.3 2.5 8.5 The seed oilsamples also contained 0.1% C14:0; 0.1-0.2% C16:3; 0.0-0.1% of each ofC20:1Δ13, C20:3ω6 and C20:4ω6; 0.3-0.4% C22:0; no C22:1 and C22:2ω6;0.2% C24:0 and 0.2-0.4% C24:1.

TABLE 23 Fatty acid composition of seed oil from T2 single seeds of B.juncea transformed with the 1-DNA from GA7-modB. Code C16:0 16:1d9 16:3C18:0 C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3 C20:0 JT-1-4-19 4.4 03 0.21.7 36.3 2.9 8.3 0.5 22.0 0.5 JT-1-4-19 5.6 0.4 0.1 1.9 39.1 3.1 8.4 0.418.9 0.6 JT-1-4-19 5.5 0.4 0.2 1.8 42.3 3.2 9.9 0.3 24.0 0.6 JT-1-4-194.7 0.3 0.1 2.3 42.1 2.8 10.0 0.2 27.1 0.6 JT-1-4-19 5.6 0.4 0.1 1.536.8 3.7 9.4 0.3 19.6 0.5 JT-1-4-19 4.6 0.3 0.1 1.7 36.3 2.7 7.2 0.322.6 0.5 JT-1-4-19 4.9 0.3 0.1 1.8 38.3 3.1 7.4 0.3 20.2 0.5 JT-1-4-194.7 0.3 0.1 1.7 36.2 3.0 8.2 0.4 20.9 0.5 JT-1-4-19 4.8 0.3 0.1 2.2 41.03.0 9.8 0.2 27.0 0.5 JT-1-4-19 5.8 0.5 0.1 1.7 36.6 3.7 9.1 0.3 21.3 0.6JT-1-4-19 4.8 0.4 0.1 2.1 47.1 2.9 7.4 0.2 23.9 0.6 JT-1-4-19 5.1 0.40.1 1.7 37.4 3.3 7.7 0.3 20.7 0.6 JT-1-4-19 4.7 0.3 0.1 1.8 37.3 2.7 7.90.4 20.6 0.5 JT-1-4-19 4.9 0.3 0.2 2.0 37.9 3.0 7.1 0.4 20.1 0.5JT-1-4-19 4.7 0.3 0.1 1.6 35.7 3.2 6.9 0.3 22.4 0.5 JT-1-4-34 4.7 0.40.1 1.8 37.6 3.4 7.8 0.3 23.7 0.5 JT-1-4-34 5.3 0.4 0.1 1.6 35.3 3.5 8.10.5 21.1 0.5 JT-1-4-34 4.9 0.3 0.1 1.7 39.4 3.3 7.7 0.3 21.1 0.5JT-1-4-34 5.0 0.3 0.1 1.8 38.5 3.1 7.8 0.4 20.5 0.5 JT-1-4-34 5.1 0.30.1 1.8 39.5 2.9 9.0 0.2 22.2 0.6 JT-1-4-34 4.8 0.3 0.1 1.8 38.2 3.2 7.80.4 21.1 0.5 JT-1-4-34 5.0 0.3 0.1 2.0 39.7 2.9 7.9 0.4 20.2 0.5JT-1-4-34 4.7 0.3 0.1 1.6 36.0 3.3 8.3 0.3 23.7 0.5 JT-1-4-34 6.2 0.50.2 2.1 32.0 4.4 7.2 0.6 19.4 0.6 Code 18:4 C20:1d11 C20:2n6 C20:3n6C20:4n6 C20:3n3 C22:0 20:4n3 20:5n3 C22:2n6 JT-1-4-19 l.4 1.2 0.1 0.00.0 0.4 0.3 4.2 0.6 0.0 JT-1-4-19 1.2 1.3 0.1 0.0 0.0 0.5 0.3 2.5 0.40.0 JT-1-4-19 5.9 1.5 0.2 0.0 0.0 0.2 0.4 0.5 0.0 0.0 JT-1-4-19 4.1 1.60.2 0.0 0.0 0.2 0.3 0.5 0.0 0.0 JT-1-4-19 0.6 1.4 0.2 0.0 0.0 1.4 0.31.9 0.3 0.0 JT-1-4-19 1.0 1.5 0.1 0.0 0.0 0.7 0.3 2.1 0.3 0.0 JT-1-4-190.8 1.3 0.1 0.0 0.0 0.8 0.3 2.7 0.5 0.0 JT-1-4-19 0.7 1.3 0.2 0.0 0.00.9 0.3 2.9 0.5 0.0 JT-1-4-19 4.2 1.8 0.2 0.0 0.0 0.3 0.3 0.5 0.0 0.0JT-1-4-19 0.9 1.4 0.2 0.0 0.0 0.8 0.3 1.5 0.3 0.0 JT-1-4-19 4.8 1.7 0.10.0 0.0 0.2 0.3 0.5 0.0 0.0 JT-1-4-19 0.9 1.4 0.1 0.0 0.0 0.8 0.3 2.50.4 0.0 JT-1-4-19 1.1 1.3 0.1 0.0 0.0 0.5 0.3 4.3 0.6 0.0 JT-1-4-19 1.11.3 0.1 0.0 0.0 0.6 0.3 4.1 0.5 0.0 JT-1-4-19 0.7 1.4 0.1 0.0 0.0 1.30.3 3.0 0.5 0.0 JT-1-4-34 0.6 1.5 0.2 0.0 0.0 1.2 0.2 1.7 0.3 0.0JT-1-4-34 0.8 1.2 0.1 0.0 0.0 0.7 0.3 3.1 0.5 0.0 JT-1-4-34 0.7 1.4 0.20.0 0.0 0.8 0.3 2.0 0.3 0.0 JT-1-4-34 0.8 1.3 0.2 0.0 0.0 0.8 0.2 2.30.3 0.0 JT-1-4-34 0.6 1.5 0.2 0.0 0.0 0.8 0.3 1.7 0.2 0.0 JT-1-4-34 0.71.4 0.2 0.0 0.0 0.7 0.3 2.1 0.4 0.0 JT-1-4-34 0.7 1.3 0.1 0.0 0.0 0.70.3 2.3 0.3 0.0 JT-1-4-34 0.6 1.5 0.2 0.0 0.0 1.2 0.3 1.7 0.3 0.0JT-1-4-34 1.2 1.2 0.2 0.0 0.0 0.6 0.4 2.2 0.5 0.0 Code C22:3n3 C24:022:5n6 22:4n3? C24:1 22:5n3 C22:6n3 JT-1-4-19 0.1 0.1 0.0 1.8 0.3 12.10.0 JT-1-4-19 0.1 0.1 0.0 1.5 0.4 12.6 0.0 JT-1-4-19 0.0 0.2 0.0 0.4 0.41.5 0.0 JT-1-4-19 0.1 0.2 0.0 0.6 0.3 1.4 0.0 JT-1-4-19 0.2 0.2 0.0 1.60.4 13.1 0.0 JT-1-4-19 0.1 0.2 0.0 2.2 0.3 14.4 0.0 JT-1-4-19 0.2 0.20.0 1.7 0.3 13.7 0.0 JT-1-4-19 0.2 0.2 0.0 2.0 0.3 14.2 0.0 JT-1-4-190.1 0.2 0.0 0.7 0.3 2.2 0.0 JT-1-4-19 0.1 0.2 0.0 1..2 0.4 12.7 0.0JT-1-4-19 0.0 0.2 0.0 0.5 0.3 1.5 0.0 JT-1-4-19 0.1 0.2 0.0 1.6 0.4 13.60.0 JT-1-4-19 0.1 0.1 0.0 2.2 0.3 12.3 0.0 JT-1-4-19 0.1 0.1 0.0 2.1 0.312.6 0.0 JT-1-4-19 0.2 0.1 0.0 1.9 0.3 14.0 0.0 JT-1-4-34 0.2 0.1 0.01.8 0.3 11.4 0.0 JT-1-4-34 0.2 0.1 0.0 1.9 0.3 13.9 0.0 JT-1-4-34 0.20.1 0.0 1.7 0.3 12.3 0.0 JT-1-4-34 0.2 0.1 0.0 2.0 0.3 13.1 0.0JT-1-4-34 0.1 0.2 0.0 1.6 0.3 10.2 0.0 JT-1-4-34 0.2 0.1 0.0 1.7 0.313.3 0.0 JT-1-4-34 0.2 0.1 0.0 1.9 0.3 12.2 0.0 JT-1-4-34 0.2 0.1 0.01.8 0.3 12.7 0.0 JT-1-4-34 0.3 0.2 0.0 1.6 0.4 17.6 0.0

TABLE 24 Fatty acid composition of seed oil from T3 single seeds of B.juncea transformed with the T-DNA from GA7-modB. C16:0 16:1d9 16:3 C18:0C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3 C20:0 18:4 C20:1d11 20:1d13 C20:2n6JT-1-4-34-11 4.8 0.4 0.1 2.8 38.4 3.7 5.7 0.4 18.0 0.7 1.0 1.5 0.1 0.1JT-1-4-34-11 4.3 0.4 0.1 3.0 43.3 3.6 5.2 0.2 18.5 0.7 0.8 1.7 0.1 0.1JT-1-4-34-11 4.6 0.4 0.1 2.8 33.1 4.1 5.1 0.4 18.5 0.7 1.2 1.4 0.1 0.1JT-1-4-34-11 4.5 0.4 0.1 2.9 39.5 3.3 6.3 0.4 18.5 0.8 1.2 1.5 0.1 0.1JT-1-4-34-11 4.9 0.5 0.2 2.8 32.2 3.9 4.7 0.3 20.7 0.8 1.2 1.4 0.1 0.2JT-1-4-34-11 4.3 0.3 0.1 3.0 38.1 3.2 5.8 0.3 19.4 0.7 1.1 1.5 0.1 0.1JT-1-4-34-11 5.4 0.5 0.2 3.2 29.3 4.0 4.6 0.4 18.6 0.9 1.7 1.3 0.1 0.1JT-1-4-34-11 5.2 0.5 0.2 3.7 34.5 4.1 4.5 0.3 17.2 1.0 1.4 1.4 0.1 0.1JT-1-4-34-11 5.3 0.5 0.1 3.4 33.4 3.7 4.6 0.3 17.6 0.9 1.7 1.2 0.1 0.1JT-1-4-34-11 4.6 0.4 0.1 3.0 39.5 3.5 5.1 0.3 17.8 0.8 0.8 1.6 0.1 0.1JT-1-4-34-11 4.3 0.4 0.1 3.1 41.7 3.5 5.6 0.2 19.0 0.7 0.9 1.6 0.1 0.1JT-1-4-34-11 4.8 0.5 0.2 2.8 33.8 4.0 5.3 0.4 18.2 0.7 1.4 1.3 0.1 0.1JT-1-4-34-11 4.4 0.4 0.1 3.5 40.3 3.5 5.2 0.2 19.1 0.7 1.0 1.5 0.1 0.1JT-1-4-34-11 4.8 0.4 0.1 3.2 36.1 3.7 5.9 0.3 19.9 0.7 1.4 1.3 0.1 0.1JT-1-4-34-11 4.0 0.3 0.1 2.8 37.2 3.2 4.9 0.3 19.6 0.8 0.9 1.6 0.1 0.1JT-1-4-34-11 4.5 0.4 0.1 3.8 36.7 3.2 4.5 0.2 19.0 0.9 1.1 1.4 0.1 0.1JT-1-4-34-11 5.2 0.4 0.2 2.8 27.8 3.7 5.3 0.5 18.3 0.8 1.7 1.3 0.1 0.1JT-1-4-34-11 5.4 0.6 0.2 2.8 31.7 4.1 4.6 0.3 18.5 0.8 1.3 1.3 0.1 0.1JT-1-4-34-11 6.4 0.6 0.1 2.7 30.3 3.5 4.1 0.4 16.1 0.8 2.1 1.1 0.1 0.1JT-1-4-34-11 4.3 0.3 0.1 3.2 39.2 3.3 5.7 0.2 20.1 0.7 0.9 1.6 0.1 0.1JT-1-4-34-11 4.2 0.4 0.1 2.3 39.9 3.9 5.9 0.3 20.2 0.6 0.9 1.3 0.1 0.1JT-1-4-34-11 4.4 0.4 0.1 2.7 38.8 3.9 5.7 0.4 18.5 0.6 1.3 1.3 0.1 0.1JT-1-4-34-11 4.2 0.4 0.1 2.0 42.4 3.6 6.0 0.4 19.1 0.5 1.0 1.6 0.1 0.1C20:3n6 C20:4n6 C20:3n3 C22:0 20:4n3 20:5n3 C22:2n6 C22:3n3 22:5n622:4n3 C24:1 22:5n3 C22:6n3 JT-1-4-34-11 0.0 0.0 1.1 0.3 1.4 0.4 0.0 0.30.0 1.4 0.5 16.3 0.0 JT-1-4-34-11 0.0 0.0 1.4 0.3 1.2 0.3 0.0 0.2 0.01.2 0.3 12.4 0.0 JT-1-4-34-11 0.0 0.0 1.1 0.3 1.6 0.5 0.0 0.3 0.0 1.40.4 20.8 0.0 JT-1-4-34-11 0.0 0.0 1.0 0.3 1.7 0.3 0.0 0.2 0.0 1.8 0.314.2 0.0 JT-1-4-34-11 0.0 0.0 2.0 0.3 1.4 0.5 0.0 0.3 0.0 1.2 0.4 19.40.0 JT-1-4-34-11 0.0 0.0 1.2 0.3 1.5 0.4 0.0 0.2 0.0 1.3 0.4 16.0 0.0JT-1-4-34-11 0.0 0.0 1.2 0.4 1.6 0.7 0.0 0.3 0.0 1.4 0.5 22.9 0.0JT-1-4-34-11 0.0 0.0 1.5 0.4 1.4 0.6 0.0 0.3 0.0 1.2 0.5 19.4 0.0JT-1-4-34-11 0.0 0.0 1.1 0.4 1.5 0.6 0.0 0.2 0.0 1.2 0.5 20.7 0.0JT-1-4-34-11 0.0 0.0 1.4 0.4 1.3 0.4 0.0 0.3 0.0 1.3 0.4 16.1 0.0JT-1-4-34-11 0.0 0.0 1.3 0.3 1.4 0.3 0.0 0.2 0.0 1.5 0.3 12.7 0.0JT-1-4-34-11 0.0 0.0 1.2 0.3 1.6 0.6 0.0 0.3 0.0 1.3 0.4 20.1 0.0JT-1-4-34-11 0.0 0.0 1.6 0.3 1.4 0.4 0.0 0.2 0.0 1.4 0.3 13.8 0.0JT-1-4-34-11 0.0 0.0 1.1 0.3 1.9 0.5 0.0 0.2 0.0 1.7 0.3 15.4 0.0JT-1-4-34-11 0.0 0.0 1.5 0.4 1.3 0.5 0.0 0.3 0.0 1.1 0.4 17.9 0.0JT-1-4-34-11 0.0 0.0 1.8 0.4 1.2 0.5 0.0 0.2 0.0 1.0 0.5 17.8 0.0JT-1-4-34-11 0.0 0.0 1.0 0.4 1.9 0.7 0.0 0.3 0.0 1.7 0.5 24.7 0.0JT-1-4-34-11 0.0 0.0 1.4 0.4 1.4 0.6 0.0 0.2 0.0 1.3 0.4 21.8 0.0JT-1-4-34-11 0.0 0.0 0.9 0.4 1.4 0.7 0.0 0.2 0.0 1.1 0.5 25.8 0.0JT-1-4-34-11 0.0 0.0 1.7 0.3 1.3 0.3 0.0 0.2 0.0 1.3 0.3 14.1 0.0JT-1-4-34-11 0.0 0.0 1.1 0.3 1.5 0.3 0.0 0.3 0.0 1.8 0.4 13.3 0.0JT-1-4-34-11 0.0 0.0 0.8 0.3 1.8 0.4 0.0 0.3 0.0 1.7 0.3 15.6 0.0JT-1-4-34-11 0.0 0.0 0.9 0.2 1.5 0.3 0.0 0.3 0.0 1.6 0.0 12.9 0.0

Example 10. Further Analysis of Transformed Plants and Field Trials

Southern blot hybridisation analysis was carried out on selected T2 B.napus plants transformed with the T-DNA from the GA7-modB construct. DNAextracted from samples of plant tissue were digested with severalrestriction enzymes for the Southern blot hybridisation analysis. Aradioactive probe corresponding to part of the T-DNA was hybridised tothe blots, which were washed under stringent conditions, and the blotsexposed to film to detect hybridising bands. Some of the samplesexhibited single hybridising bands for each of the restriction digests,corresponding to single T-DNA insertions in the plants, while othersshowed two bands and others again showed multiple T-DNA bands,corresponding to 4 to 6 insertions. The number of hybridising bandsobserved by Southern Blot analysis correlated well with the T-DNA copynumber in the transgenic plants as determined by the digital PCR method,up to a copy number of about 3 or 4. At higher copy numbers than about5, the digital PCR method was less reliable.

Some of the selected lines were used as pollen donors in crosses with aseries of about 30 different B. napus varieties of different geneticbackgrounds. Further back-crosses are carried out to demonstrate whetherthe multiple T-DNA insertions are genetic linked or not, and allowingsegregation of genetically-unlinked transgenic loci. Thereby, linescontaining single transgenic loci are selected.

Single-primer PCR reactions are carried out on the transgenic lines,using primers adjacent to the left- and right-borders of the T-DNA, andany lines that show the presence of inverted repeats of the T-DNAs arediscarded.

Several of the transgenic lines showed delayed flowering, while othershad reduced seed-set and therefore reduced seed yield per plant aftergrowth in the glasshouse, consistent with a reduced male or femalefertility. Flower morphology was examined in these plants and it wasobserved that in some cases, dehiscence and release of pollen from theanthers was delayed so that styles had elongated before dehiscenceoccurred, thereby distancing the anthers from the stigmas. Fullfertility could be restored by artificial pollination. Furthermore,pollen viability at dehiscence was determined by staining with the vitalstains FDA and PI (Example 1) and was shown to be reduced in some of thelines, whereas in most of the transgenic lines, pollen viability wasabout 100% as in the wild type controls. As a further test for apossible cause of the reduced seed yield in some plants, the fatty acidcontent and composition of flower buds including the anthers andstigmas/styles of some T3 and T4 plants was tested. No DHA was detectedin the extracted lipids, indicating that the genes in the geneticconstruct were not expressed in the flower buds during plantdevelopment, and ruling this out as a cause of the reduced seed yield.

The oil content was measured by NMR and the DHA level in the total fattyacid content was determined for T2 seeds. Transgenic lines having lessthan 6% DHA were discarded. T-DNA copy number in leaf samples fromplants of the T1, T2 and T3 generations were determined by the digitalPCR method (Example 1).

Selected T3 and T4 seed lots were sown in the field at two sites inVictoria, Australia, each in 10 m rows at a sowing density of about 10seeds/m. The selected seed lots included a B003-5-14 derived line whichshowed pooled seed DHA levels of about 8-11% and individual T2 seed DHAlevels of up to about 19%, with a TO plant T-DNA copy number of 3. Theselected seed lots also included B0050-27 derived lines which had shownT2 seed DHA levels in excess of 20%, and a T2 plant T-DNA copy number of1 or 2. Seeds sown in the field germinated and plantlets emerged at thesame rate as the wild-type seeds. Plants grown from most, but not all,of the sown seed lots were phenotypically normal, for example hadmorphology, growth rate, plant height, male and female fertility, pollenviability (100%), seed set, silique size and morphology that wasessentially the same as the wild-type control plants grown under thesame conditions. Seed yield per plant was similar to that of wild-typecontrols grown under the same conditions. Other seed samples were sownin larger areas to bulk-up the selected transgenic lines. The total DHAcontent in harvested seeds was at least 30 mg/g seed.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from AU 2013905033 filed 18 Dec.2013, and AU 2014902471 filed 27 Jun. 2014, the entire contents of bothof which are incorporated herein by reference. All publicationsdiscussed and/or referenced herein are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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The invention claimed is:
 1. A process for producing extracted Brassicanapus lipid, comprising the steps of: i) obtaining Brassica napus seedcomprising extractable lipid having a total fatty acid contentcomprising fatty acids in an esterified form, the fatty acids comprisingoleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid(LA), ω3 fatty acids which comprise α-linolenic acid (ALA), stearidonicacid (SDA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA),eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein thelevel of DHA in the total fatty acid content of extractable lipid in theBrassica napus seed is between 20.1% and 30%, and ii) extracting lipidfrom the Brassica napus seed, to thereby produce extracted Brassicanapus lipid.
 2. The process of claim 1 which further comprises treatingthe extracted Brassica napus lipid to increase the level of DHA as apercentage of the total fatty acid content, wherein the treatmentcomprises one or more of fractionation, distillation ortransesterification.
 3. The process of claim 2 which comprises theproduction of methyl- or ethyl-esters of DHA.
 4. The process of claim 1,wherein the level of oleic acid in the total fatty acid content of theextractable lipid is between 30% and 60%.
 5. The process of claim 1,wherein the level of LA in the total fatty acid content of theextractable lipid is between 4% and 17%.
 6. The process of claim 1,wherein the level of ALA in the total fatty acid content of theextractable lipid is between 7% and 40%.
 7. The process of claim 1,wherein the level of EPA in the total fatty acid content of theextractable lipid is less than 4%.
 8. The process of claim 1, whereinthe level of DPA in the total fatty acid content of the extractablelipid is less than 3%.
 9. The process of claim 1, wherein the level ofDHA in the total fatty acid content of the extractable lipid is between20.1% and 25%.
 10. The process of claim 1, wherein the level of totalsaturated fatty acids in the total fatty acid content of the extractablelipid is between 6% and 12%.
 11. The process of claim 1, wherein theratio of total ω6 fatty acids:total ω3 fatty acids in the total fattyacid content of the extractable lipid is between 0.1 and
 1. 12. Theprocess of claim 1, wherein in the extractable lipid at least 80% of theDHA esterified in the form of TAG is in the sn-1 or sn-3 position of theTAG.
 13. A process for producing extracted Brassica napus lipid,comprising the steps of: i) obtaining Brassica napus seed comprisingextractable lipid having a total fatty acid content comprising fattyacids in an esterified form, the fatty acids comprising oleic acid,palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3fatty acids which comprise α-linolenic acid (ALA), stearidonic acid(SDA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA),eicosapentaenoic acid (EPA) and eicosatetraenoic acid (ETA), wherein thelevel of DHA in the total fatty acid content of extractable lipid in theBrassica napus seed is between 30% and 35%, and ii) extracting lipidfrom the Brassica napus seed to thereby produce extracted Brassica napuslipid.
 14. The process of claim 13 which further comprises treating theextracted Brassica napus lipid to increase the level of DHA as apercentage of the total fatty acid content, wherein the treatmentcomprises one or more of fractionation, distillation ortransesterification.
 15. The process of claim 14 which comprises theproduction of methyl- or ethyl-esters of DHA.
 16. The process of claim13, wherein the level of EPA in the total fatty acid content of theextractable lipid is less than 4%.
 17. The process of claim 13, whereinthe level of DPA in the total fatty acid content of the extractablelipid is less than 3%.
 18. The process of claim 13, wherein the level oftotal saturated fatty acids in the total fatty acid content of theextractable lipid is between 6% and 12%.
 19. The process of claim 13,wherein the ratio of total ω6 fatty acids:total ω3 fatty acids in thetotal fatty acid content of the extractable lipid is between 0.1 and 1.20. The process of claim 13, wherein in the extractable lipid at least80% of the DHA esterified in the form of TAG is in the sn-1 or sn-3position of the TAG.